http://measurebiology.org/w/api.php?action=feedcontributions&user=Steven+Nagle&feedformat=atomCourse Wiki - User contributions [en]2024-03-28T21:44:26ZUser contributionsMediaWiki 1.22.3http://measurebiology.org/wiki/Optical_Microscopy_Part_1:_Brightfield_MicroscopyOptical Microscopy Part 1: Brightfield Microscopy2016-01-11T21:10:49Z<p>Steven Nagle: /* Practice */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:Optical Microscopy Lab]]<br />
{{Template:20.309}}<br />
<br />
<blockquote><br />
<div><br />
''These I mention, that I may excite the World to enquire a little farther into the improvement of Sciences, and not think that either they or their predecessors have attained the utmost perfections of any one part of knowledge, and to throw off that lazy and pernicious principle, of being contented to know as much as their Fathers, Grandfathers, or great Grandfathers ever did, and to think they know enough, because they know somewhat more than the generality of the World besides:…Let us see what the improvement of Instruments can produce.''<br />
<blockquote><br />
''&mdash;Animadversions on the Machina Coelestis of Johannes Hevelius, 1674''<br />
</blockquote><br />
</div><br />
</blockquote><br />
<br />
<blockquote><br />
<div><br />
''Don't you just '''buy''' a [http://www.historycommons.org/context.jsp?item=a043074editedtranscripts [expletive deleted]] microscope?''<br />
<br />
<blockquote><br />
''&mdash;[http://web.mit.edu/fll/www/events/AforE/images/AEWinner1.jpg Anonymous 20.309 student], Fall 2007''<br />
</blockquote><br />
</div><br />
</blockquote><br />
<br />
==Overview==<br />
In the first week of the microscopy lab, you will construct a brightfield microscope.<br />
<br />
====Background materials and references==== <br />
<br />
The following online materials provide useful background for this part of the microscopy lab.<br />
<br />
* [[Geometrical optics and ray tracing]]<br />
* [[Physical optics and resolution]]<br />
* [https://stellar.mit.edu/S/course/20/fa13/20.309/materials.html Lectures 1 through 9 of the 20.309 class]<br />
* From [http://www.microscopyu.com Nikon MicroscopyU]<br />
** [http://www.microscopyu.com/articles/formulas/formulasconjugate.html Conjugate planes in optical microscopy] (includes transmitted and reflected (epi) illumination)<br />
** [http://www.microscopyu.com/articles/formulas/formulasri.html Snell's law]<br />
** [http://www.microscopyu.com/articles/formulas/formulasresolution.html Resolution]<br />
<br />
===Microscope block diagram===<br />
<br />
[[Image:20.309 130911 YourMicroscope.png|center|thumb|400px|20.309 microscope block diagram]]<br />
<br />
==Optical construction==<br />
Once you have settled on an arrangement of lenses and filters, the next challenge is to construct a system that will hold all the optics in their proper places and allow you to precisely locate a sample in front of the objective. Some of the optics must be very placed very accurately, while others don't matter so much. The position and angle of some components must be adjustable. The structure should be very rigid so that vibration does not degrade your images. <br />
<br />
There are several systems for optical construction available based on rails, posts, cages, tubes, and all manner of little, metallic bits. The 20.309 microscope is constructed chiefly from cage and lens tube components made by a company called [http://www.thorlabs.com/ ThorLabs]. Understanding how all of the components in the catalog work together is daunting. Ask about any components that perplex you.<br />
<br />
====Lenses====<br />
<br />
Plano-convex spherical lenses are available with focal lengths of 25, 50, 75, 100, 125, 150, 175, and 200 mm. Plano-concave lenses with focal lengths of -30 and -50 are also available. It is best to mount most optics in short (''e.g.'' 0.5") lens tubes. It is acceptable to mount a lens between the end of a tube and a tube ring or between two tube rings. In most cases, the convex side of the lens faces toward the collimated beam; the planar side goes toward the convergent rays. <br />
<br />
* ''Tip:'' Verify all optics before you use them by determining the focal length with a ruler. Use the ceiling fluorescent lamps as a light source and measuring the exact distance between the lens(es) assessed and the lamp's image. Can you imagine a simple rig to evaluate negative focal lengths (of plano-concave lenses for instance)?<br />
* ''Tip:'' As you install lenses into your microscope, put a piece of tape on the lens tube showing focal length and orientation. This will help you both during construction and put-away. Save the lens storage boxes and return components to the correct boxes when you are done.<br />
* Handle lenses only by the edges. If a lens is dirty, first remove grit with a blast of clean air or CO<sub>2</sub>. Clean the lens by wiping with a folded piece of lens paper wetted with a drop of methanol. (Do not touch the part of the tissue you use for cleaning with your fingers.) In some cases, it may be helpful to hold the folded lens tissue in a hemostat. Ask an instructor if you need help.<br />
<br />
====Objective lenses====<br />
<br />
Please see the Nikon [http://www.microscopyu.com/articles/optics/objectiveintro.html Introduction to Microscope Objectives] at their excellent [http://www.microscopyu.com/index.html MicroscopyU] website.<br />
<br />
There are three objective lenses available in the lab: a 10×, a 40×, and a 100×. All of these are designed to use a 200 mm tube lens to form an image on the camera. An adapter ring converts the objective mounting threads to the SM1 threads used by the lens tube system.<br />
<br />
[[Image:20.309_130813_SimpleMicroscopeDiagram.png|center|thumb|400px|The reference tube length for the Nikon objectives we will use is 200 mm. A 200 mm lens, placed 200 mm from the CCD, will produce the rated magnification M.]]<br />
* ''Working distance'' (WD) is the distance between the front objective lens surface and the cover slip, and so it is also approximately the distance to the front focal plane. In order to focus an image at the back focal plane of the tube lens, i.e., on the CCD array, the sample plane must coincide with the front focal plane in a 4f microscope arrangement. The stage is added to hold the sample in this plane.<br />
* Note that the 100× objective is designed to be used with immersion oil, which provides an optical medium of pre-determined refractive index (''n'' = 1.5). When using the 100× objective, place a drop of oil on it. Bring the drop in contact with the slide cover glass. After use, clean off excess oil by wicking it away with lens paper. Do not put samples away dirty. It is not necessary to use immersion oil for thin samples such as the Air Force Target or Ronchi Ruling.<br />
* Note that the back focal plane (BFP) of the objective does not necessarily coincide with the rear of the objective housing. In fact, for the Nikon 40x objective the BFP is close to the blue ring. You will find its actual location when aligning the laser path in Part 2. The 200 mm distance labeled between the back of the objective housing and the tube lens is a recommendation from Nikon to enable optimal imaging. For details on the importance and origin of this distance please ask an instructor.<br />
<br />
====Sample stage====<br />
<br />
A precision Newport X/Y/Z stage<ref>[http://www.newport.com/562-Series-ULTRAlign-Precision-Multi-Axis-Positio/140089/1033/catalog.aspx Precision Newport X/Y/Z stages]</ref> with a sample holder mounted on a post, or a Thorlabs Max312D stage, also with a sample holder, is available at each lab station. The Newport stage setup is top-heavy. Avoid accidents by ensuring that the post base is always attached to an optical breadboard or table. Leave the stage at the lab station when you are done with it. For the Thorlabs stages, it is still a good idea to bolt them down so that your area of interest (AOI) stays in your microscope field of view (FOV).<br />
<br />
All stage axes have limited adjustment range, especially the Thorlabs stages. To deal with this, it is best to leave the stage base bolts and sample holder bolts loose and move the sample holder in x, y and z to roughly find your AOI. Once you are on or near your AOI, tighten the bolts and use the micrometers to center your image. One trick here is to get the z clamped first, then deal with x and y.<br />
<br />
====CCD camera====<br />
<br />
The microscope you will build does not have an eyepiece for direct visual observation. Instead, images will be captured with a CCD camera<ref>[http://www.alliedvisiontec.com/emea/products/cameras/gigabit-ethernet/manta/g-032bc.html Allied Manta G032B]</ref>. Its monochrome (black and white) sensor contains a grid of 656×492 square pixels that measure 7.4 μm on a side. An adapter ring converts the C-mount thread on the camera to SM1.<br />
<br />
==Microscope construction==<br />
<br />
===Design===<br />
<br />
Sketch out a rough design for your microscope on paper. Begin with the bright field illumination path.<br />
* Some elements must be positioned precise distances apart; other distances are not critical. Use ray-tracing to determine when this is the case. Which distances in your bright-field microscope will be critical? Which will be forgiving or unessential? Which will change with each objective lens (10×, 40× and 100×)?<br />
* Which sections of the light path can be open (strut-based structure)? Which would better enclosed (Thorlabs tubes)?<br />
* In what way will the illumination LED color affect your design? your results?<br />
* Which lens will you use between the LED and the sample for bright-field transmitted light imaging?<br />
<br />
===Practice===<br />
<br />
* Please do not remove parts from the example microscope.<br />
* On a 1' x 2' x <sup>1</sup>/<sub>2</sub>" optical breadboard, build a bright-field imaging microscope, using the provided LED as a light source, CCD camera as a light detector, and the rigid mounting components and lenses at your disposal.<br />
* Even though you're first focusing on the bright-field imaging leg of your microscope, take into consideration some requirements pertinent to the fluorescence imaging elements you'll add to your system next week:<br />
** Reproduce the general layout of the example microscope: it grants compactness and allows your device to be a stand-alone breadboard-transportable microscope. <br />
*** Set the distance between the top of the breadboard and the top surface of the upper LCP01 to '''13.5 cm'''. It is important to ensure your construction is compatible with either of the two distinct stage mounting platforms available in the 20.309 lab (either Newport or Thorlabs model). If you find it inconvenient to measure this, there is a Handy Scope Height Thingama-jig floating around the lab. Ask your instructor(s). Also, note that the stages are very expensive; always lift them from the bottom.<br />
*** Align the vertical Thorlabs P14 (1.5" diameter mounting post) with a breadboard hole that is 11 positions from a short side and 5 positions from a long side. This allows enough free space on the breadboard such that either the Newport or the Thorlabs stages may be utilized. (Note that the picture is in error as the P14 is only 9 positions from a short side of the breadboard.)<br />
[[Image:130814_Microscope_All.jpg|center|thumb|400px|The general layout of the 20.309 microscope is compact and stand-alone; it fits and can be transported onto a breadboard]]<br />
** Do insert the C6W cage cube that will later hold the dichroic mirror on while fluorescence imaging will rely. Be sure to keep the mounting struts fully recessed in the cube walls; their ends should not stick out, they would otherwise hinder maneuvers with dichroic-holding kinematic plate!<br />
[[Image:130816_CageCube.png|center|thumb|400px|The mounting struts should remain recessed within the cage cube walls.]]<br />
<br />
* Verify the focal length of the lenses you selected. If you find an optic in the wrong box: identify the optic and replace it in the correct box or label the box correctly. (Ask an instructor if you can't find the right box. There are many boxes near the wire spools behind you as you stand at the wet bench.)<br />
* Check all your lenses for cleanliness before you use them. You'll save yourself some troubleshooting time and effort down the road!<br />
* Make sure all your components are "leveled" (horizontal, not slanted).<br />
* Use tube rings (and never an SM1T2, SM1V01, or SM1V05) to mount optics in lens tubes.<br />
* Use adjustable mounting components in front of the CCD camera so you can optimize and fine-tune the camera positing with respect to the imaging lens ''L2''. Beware: never use an SM1T2 coupler without a locking ring &mdash; they are very difficult to remove if they are tightened against a lens tube or tube ring. Also put a quick-connect in your design such that the camera CCD will end up 200 mm from the back focal plane of the objective. Remember that the CCD is recessed inside the opening of the camera.<br />
[[Image:20.309 130816 CCD QuickConnect.png|center|thumb|400px|Adjustable Thorlabs SM1V05 and SM1T2 connectors precede the quick-connect union to the CCD camera.]]<br />
* Use only three cage rails to connect the C6W cage cube and the KCB containing the last silver mirror before the CCD camera, so you can easily take in and out the barrier filter (BF) that will later aid fluorescence-mode microscopy. Always place two rails at the top so that an alignment target can be hung if needed (the benefit will become more clear in Part 2).<br />
<br />
[[Image:20.309 130816 BarrierFilterSpace.png|center|thumb|250px| Insertion and removal of optical components is facilitated by a three-strut-only link. ]]<br />
<br />
* The Nikon objective lenses are designed to be paired with a 200 mm tube lens.<br />
* Assume that the objectives behave as ideal plano-convex lenses.<br />
* Fine focusing will be achieved by adjusting the height of the sample stage.<br />
* ''Tip:'' Throughout the optical microscopy lab, start the alignment with a 10× objective and then progress to 40× and 100×.<br />
* A red or a blue LED illuminator can be used for bright-field transmitted light imaging. On one hand a blue LED yields a better bright-field resolution, however bright-field resolution is not usually critical in this lab. On the other hand, a red LED allows simultaneous fluorescent and bright-field imaging in Parts 2, 3 and 4, and this can be quite useful when trying to bring a fluorescent sample into focus.<br />
** Each group will receive their own LED. Please ask an instructor if you cannot find one.<br />
{{Template:Safety Warning|message=Double check your wiring before powering the LED. The LED can be damaged by excessive current. Limit the driving current to 0.5 A to protect the LED.}}<br />
<br />
==Magnification measurement==<br />
[[Image:20.309_130813_BrightFieldExampleImages.png|right|thumb|Example images included by past students in their Week 1 report: (top) Air Force target, (center) Silica spheres and dust, (bottom) Ronchi Ruling]]<br />
Measuring the magnification of your microscope is a good way to verify that your instrument is functioning well. You should measure the magnification of any microscope you plan to use for making quantitative measurements of size. Use the measured value in your calculations, not the number printed on the objective. Consider the uncertainty in your measurement.<br />
<br />
# Use MATLAB's Image Acquisition Tool to view a live display and record images<br />
#* Launch Matlab and type <tt>imaqtool</tt>. An Image Acquisition Tool window should fill the screen.<br />
#* Select the "Mono12" mode of the "Manta_G-032B (gentl-1)" camera in the Hardware Browser pane.<br />
#* Once it behaves, this setting will configure the camera to produce 12-bit, monochrome images. In this mode, the intensity of each pixel in the image will be represented by 12 binary digits, allowing a range of values from 0-4095.<br />
#* In the Acquisition Parameters pane, select the "Device Properties" tab and set "Acquisition Frame Rate Abs" to 20. This will cause the camera to take 20 complete images per second.<br />
#* Click the "Start Preview" button. The live image from the camera should appear in the Preview pane.<br />
#* If this does not produce a live image, close the window, issue the command imaqreset in the Matlab workspace. Then issue the command imaqtool again, choose the "gige-1" driver and start it as above. Regardless of whether it works, now close the window, issue the imaqreset command again, then the imaqtool command, and continue by choosing the "gentl-1" option. The drivers are, it goes without saying, a little flaky. It is best to avoid the "gige-1" option for long-running because it is more prone to hanging up than the "gentl-1" option.<br />
#* Use the "Exposure Auto" and "Exposure Time Abs" settings in the "Device Properties" tab to produce a good image. Setting "Exposure Auto" to "Once" will cause the camera to run its automatic exposure algorithm one time. This usually results in an exposure that's in the ballpark. But the automatic exposure usually does a poor job on microscopic images. Make the exposure better by changing the value in "Exposure Time Abs". The value sets the exposure time for each frame in microseconds.<br />
# Ensure that the camera's field of view is approximately centered in the objective's field of view. <br />
#* The objective has a larger FOV than the camera. Use the adjustment knobs on mirror M1 to traverse the objective's FOV horizontally and vertically. The FOV is approximately circular. Find a spot near the middle.<br />
# Measure the magnification of your microscope using the 10x, 40x, and 100x objectives.<br />
#* Start with the 10x objective and an Air Force imaging target.<br />
#** There are two styles of Air Force imaging target available in the lab. Both have sets of precisely spaced vertical and horizontal, high-contrast black/white line pairs. One version of the target indicates the size of the line pairs with a number conveniently printed near the set of lines. The number indicates how many black/white line pairs per millimeter. The other style of Air Force imaging target uses an annoying group/element designation that is explained on [[http://en.wikipedia.org/wiki/1951_USAF_resolution_test_chart this Wikipedia page]]. <br />
#** Make sure that the side of the target with the pattern on it faces the objective. Imaging through the thick glass causes distortion and many other troubles.<br />
#* Record an image of appropriately sized lines on the Air Force imaging target (with 10x and 40x objectives) and the Ronchi ruling (with the 100x objective).<br />
#** Why is it not judicious to image the Ronchi ruling with the 10x objective?<br />
#* Set "Frames per trigger" setting in the "General" tab of the Acquisition Parameters pane to 1. This setting controls how many images MATLAB will record each time you press the "Start Acquisition" button. <br />
#* Press the "Start Acquisition" in the Preview Pane. (The live preview will stop.)<br />
#* Press "Export Data..." In the dialog that comes up, select "MATLAB Workspace" in the "Data Destination" popup and type in a variable name, such as <tt>AirForce14lp10x</tt>. Data from the image you acquired will be available in the Matlab workspace.<br />
#* Switch to the MATLAB command window and type <tt>whos AirForce14lp10x</tt>. The image is represented as a 492x656 matrix of 16-bit integers.<br />
#* To display the image, use the <tt>imshow</tt> command. <br />
#** When the 12-bit numbers from the camera get transferred to the computer, they are converted to 16-bit numbers. 16-bit numbers can represent a range of values from 0-65535. This leaves a considerable portion of the number range unoccupied. Because of this, if you type <tt>imshow( AirForce14lp10x )</tt>, you will see an image that looks almost completely black. Adjust the image to fill the full range by typing <tt>imshow( 16.0037 * AirForce14lp10x )</tt>. 16.0037 equals 65535/4095. This factor maps values in the range 0-4095 to 0-65535.<br />
#* An even better way to work with images in MATLAB is to convert them to [[http://en.wikipedia.org/wiki/Double-precision_floating-point_format double precision floating point format]] straightaway. Double precision floating point numbers can represent an extremely wide range of values with high precision. Matlab includes a function for converting images, <tt>im2double</tt>. Type <tt>AirForce14lp10x = im2double( 16.0037 * AirForce14lp10x );</tt> to make the conversion and then use <tt>imshow</tt> to see the result. After the conversion to double, the range of intensities is mapped to 0-1, with 1 being full intensity and 0 completely dark.<br />
#* Use the data cursor to measure magnification<br />
#** When choosing the size of lines to image, consider the factors that influence the uncertainty of your measurement.<br />
#* Save your image as a .mat for later use in MATLAB (<tt>save AirForce14lp10x</tt>) or as a PNG image for use in your report or other programs. if you converted the image to double, the command might look something like: <tt>imwrite( im2uint16( AirForce14lp10x ), 'AirForce14lp10x.png', 'png' );</tt>.<br />
# Repeat the magnification measurement for the 40x and 100x objectives. <br />
#* With the 100x objective, you may want to substitute the Air Force target with a Ronchi Ruling, a grating with 600 line pairs per millimeter.<br />
# Calculate the FOV of the microscope using all three objectives.<br />
<br />
==Particle size measurement==<br />
[[Image:20.309_130813_BF_3p2umbeads_40x.png|right|thumb|Example image of 3.2 μm beads using the instructor microscope. Submit picture to replace this!]]<br />
<br />
Now that you know the magnification of your instrument, use it to measure the size of some microscopic objects as imaged with the 40x objective lens only. Slides with 7.2 μm, 3.2 μm and 1 μm silica microspheres are available in the lab.<br />
<br />
# Image 7.2 μm, 3.2 μm and 1 μm silica microspheres as described in the magnification measurement procedure (40x objective only). <br />
# Measure and report the average size and uncertainty of the spheres in each sample. How many spheres should you measure?<br />
<br />
==Microscope storage==<br />
<br />
During the microscopy lab, approximately seven thousand optical components will be taken from stock, assembled into microscopes, and properly returned to their assigned places. Please observe the following:<br />
* Store your microscope in one of the cubby holes in 16-336 (not in the lab). If you use one of the high shelves, get somebody to help you lift.<br />
* Keep all of the boxes for the optics you use with your instrument to simplify putting things away. <br />
* Take a blue bin to store loose items (such as lens boxes) in.<br />
* Stages, CCD cameras, neutral density filters and barrier filters stay at the lab station. Do not store these with your microscope.<br />
* Return objective lenses to the drawer when you are not using them. (Do not store them with your microscope.)<br />
* The stages are very expensive. Always lift from the bottom.<br />
* If you break something (or discover something pre-broken for you), do not return it to the component stock. Give all broken items to an instructor. You will not be penalized for breaking something, but not reporting may be looked upon less kindly.<br />
<br />
==Report outline==<br />
<br />
Find and follow all guidelines on the [[Microscopy report outline]] wiki page. <br />
<br />
{{:Optical Microscopy: Part 1 Report Outline}}<br />
<br />
{{:Optical microscopy lab wiki pages}}<br />
<br />
==References==<br />
<references /><br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/Optical_Microscopy_Part_1:_Brightfield_MicroscopyOptical Microscopy Part 1: Brightfield Microscopy2016-01-11T20:37:20Z<p>Steven Nagle: /* Objective lenses */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:Optical Microscopy Lab]]<br />
{{Template:20.309}}<br />
<br />
<blockquote><br />
<div><br />
''These I mention, that I may excite the World to enquire a little farther into the improvement of Sciences, and not think that either they or their predecessors have attained the utmost perfections of any one part of knowledge, and to throw off that lazy and pernicious principle, of being contented to know as much as their Fathers, Grandfathers, or great Grandfathers ever did, and to think they know enough, because they know somewhat more than the generality of the World besides:…Let us see what the improvement of Instruments can produce.''<br />
<blockquote><br />
''&mdash;Animadversions on the Machina Coelestis of Johannes Hevelius, 1674''<br />
</blockquote><br />
</div><br />
</blockquote><br />
<br />
<blockquote><br />
<div><br />
''Don't you just '''buy''' a [http://www.historycommons.org/context.jsp?item=a043074editedtranscripts [expletive deleted]] microscope?''<br />
<br />
<blockquote><br />
''&mdash;[http://web.mit.edu/fll/www/events/AforE/images/AEWinner1.jpg Anonymous 20.309 student], Fall 2007''<br />
</blockquote><br />
</div><br />
</blockquote><br />
<br />
==Overview==<br />
In the first week of the microscopy lab, you will construct a brightfield microscope.<br />
<br />
====Background materials and references==== <br />
<br />
The following online materials provide useful background for this part of the microscopy lab.<br />
<br />
* [[Geometrical optics and ray tracing]]<br />
* [[Physical optics and resolution]]<br />
* [https://stellar.mit.edu/S/course/20/fa13/20.309/materials.html Lectures 1 through 9 of the 20.309 class]<br />
* From [http://www.microscopyu.com Nikon MicroscopyU]<br />
** [http://www.microscopyu.com/articles/formulas/formulasconjugate.html Conjugate planes in optical microscopy] (includes transmitted and reflected (epi) illumination)<br />
** [http://www.microscopyu.com/articles/formulas/formulasri.html Snell's law]<br />
** [http://www.microscopyu.com/articles/formulas/formulasresolution.html Resolution]<br />
<br />
===Microscope block diagram===<br />
<br />
[[Image:20.309 130911 YourMicroscope.png|center|thumb|400px|20.309 microscope block diagram]]<br />
<br />
==Optical construction==<br />
Once you have settled on an arrangement of lenses and filters, the next challenge is to construct a system that will hold all the optics in their proper places and allow you to precisely locate a sample in front of the objective. Some of the optics must be very placed very accurately, while others don't matter so much. The position and angle of some components must be adjustable. The structure should be very rigid so that vibration does not degrade your images. <br />
<br />
There are several systems for optical construction available based on rails, posts, cages, tubes, and all manner of little, metallic bits. The 20.309 microscope is constructed chiefly from cage and lens tube components made by a company called [http://www.thorlabs.com/ ThorLabs]. Understanding how all of the components in the catalog work together is daunting. Ask about any components that perplex you.<br />
<br />
====Lenses====<br />
<br />
Plano-convex spherical lenses are available with focal lengths of 25, 50, 75, 100, 125, 150, 175, and 200 mm. Plano-concave lenses with focal lengths of -30 and -50 are also available. It is best to mount most optics in short (''e.g.'' 0.5") lens tubes. It is acceptable to mount a lens between the end of a tube and a tube ring or between two tube rings. In most cases, the convex side of the lens faces toward the collimated beam; the planar side goes toward the convergent rays. <br />
<br />
* ''Tip:'' Verify all optics before you use them by determining the focal length with a ruler. Use the ceiling fluorescent lamps as a light source and measuring the exact distance between the lens(es) assessed and the lamp's image. Can you imagine a simple rig to evaluate negative focal lengths (of plano-concave lenses for instance)?<br />
* ''Tip:'' As you install lenses into your microscope, put a piece of tape on the lens tube showing focal length and orientation. This will help you both during construction and put-away. Save the lens storage boxes and return components to the correct boxes when you are done.<br />
* Handle lenses only by the edges. If a lens is dirty, first remove grit with a blast of clean air or CO<sub>2</sub>. Clean the lens by wiping with a folded piece of lens paper wetted with a drop of methanol. (Do not touch the part of the tissue you use for cleaning with your fingers.) In some cases, it may be helpful to hold the folded lens tissue in a hemostat. Ask an instructor if you need help.<br />
<br />
====Objective lenses====<br />
<br />
Please see the Nikon [http://www.microscopyu.com/articles/optics/objectiveintro.html Introduction to Microscope Objectives] at their excellent [http://www.microscopyu.com/index.html MicroscopyU] website.<br />
<br />
There are three objective lenses available in the lab: a 10×, a 40×, and a 100×. All of these are designed to use a 200 mm tube lens to form an image on the camera. An adapter ring converts the objective mounting threads to the SM1 threads used by the lens tube system.<br />
<br />
[[Image:20.309_130813_SimpleMicroscopeDiagram.png|center|thumb|400px|The reference tube length for the Nikon objectives we will use is 200 mm. A 200 mm lens, placed 200 mm from the CCD, will produce the rated magnification M.]]<br />
* ''Working distance'' (WD) is the distance between the front objective lens surface and the cover slip, and so it is also approximately the distance to the front focal plane. In order to focus an image at the back focal plane of the tube lens, i.e., on the CCD array, the sample plane must coincide with the front focal plane in a 4f microscope arrangement. The stage is added to hold the sample in this plane.<br />
* Note that the 100× objective is designed to be used with immersion oil, which provides an optical medium of pre-determined refractive index (''n'' = 1.5). When using the 100× objective, place a drop of oil on it. Bring the drop in contact with the slide cover glass. After use, clean off excess oil by wicking it away with lens paper. Do not put samples away dirty. It is not necessary to use immersion oil for thin samples such as the Air Force Target or Ronchi Ruling.<br />
* Note that the back focal plane (BFP) of the objective does not necessarily coincide with the rear of the objective housing. In fact, for the Nikon 40x objective the BFP is close to the blue ring. You will find its actual location when aligning the laser path in Part 2. The 200 mm distance labeled between the back of the objective housing and the tube lens is a recommendation from Nikon to enable optimal imaging. For details on the importance and origin of this distance please ask an instructor.<br />
<br />
====Sample stage====<br />
<br />
A precision Newport X/Y/Z stage<ref>[http://www.newport.com/562-Series-ULTRAlign-Precision-Multi-Axis-Positio/140089/1033/catalog.aspx Precision Newport X/Y/Z stages]</ref> with a sample holder mounted on a post, or a Thorlabs Max312D stage, also with a sample holder, is available at each lab station. The Newport stage setup is top-heavy. Avoid accidents by ensuring that the post base is always attached to an optical breadboard or table. Leave the stage at the lab station when you are done with it. For the Thorlabs stages, it is still a good idea to bolt them down so that your area of interest (AOI) stays in your microscope field of view (FOV).<br />
<br />
All stage axes have limited adjustment range, especially the Thorlabs stages. To deal with this, it is best to leave the stage base bolts and sample holder bolts loose and move the sample holder in x, y and z to roughly find your AOI. Once you are on or near your AOI, tighten the bolts and use the micrometers to center your image. One trick here is to get the z clamped first, then deal with x and y.<br />
<br />
====CCD camera====<br />
<br />
The microscope you will build does not have an eyepiece for direct visual observation. Instead, images will be captured with a CCD camera<ref>[http://www.alliedvisiontec.com/emea/products/cameras/gigabit-ethernet/manta/g-032bc.html Allied Manta G032B]</ref>. Its monochrome (black and white) sensor contains a grid of 656×492 square pixels that measure 7.4 μm on a side. An adapter ring converts the C-mount thread on the camera to SM1.<br />
<br />
==Microscope construction==<br />
<br />
===Design===<br />
<br />
Sketch out a rough design for your microscope on paper. Begin with the bright field illumination path.<br />
* Some elements must be positioned precise distances apart; other distances are not critical. Use ray-tracing to determine when this is the case. Which distances in your bright-field microscope will be critical? Which will be forgiving or unessential? Which will change with each objective lens (10×, 40× and 100×)?<br />
* Which sections of the light path can be open (strut-based structure)? Which would better enclosed (Thorlabs tubes)?<br />
* In what way will the illumination LED color affect your design? your results?<br />
* Which lens will you use between the LED and the sample for bright-field transmitted light imaging?<br />
<br />
===Practice===<br />
<br />
* Please do not remove parts from the example microscope.<br />
* On a 1' x 2' x <sup>1</sup>/<sub>2</sub>" optical breadboard, build a bright-field imaging microscope, using the provided LED as a light source, CCD camera as a light detector, and the rigid mounting components and lenses at your disposal.<br />
* Even though you're first focusing on the bright-field imaging leg of your microscope, take into consideration some requirements pertinent to the fluorescence imaging elements you'll add to your system next week:<br />
** Reproduce the general layout of the example microscope: it grants compactness and allows your device to be a stand-alone breadboard-transportable microscope. [[Image:130814_Microscope_All.jpg|center|thumb|400px|The general layout of the 20.309 microscope is compact and stand-alone; it fits and can be transported onto a breadboard]]<br />
** Do insert the C6W cage cube that will later hold the dichroic mirror on while fluorescence imaging will rely. Be sure to keep the mounting struts fully recessed in the cube walls; their ends should not stick out, they would otherwise hinder maneuvers with dichroic-holding kinematic plate!<br />
[[Image:130816_CageCube.png|center|thumb|400px|The mounting struts should remain recessed within the cage cube walls.]]<br />
* Set the distance between the top of the breadboard and the top of the upper LCP01 to '''13.5 cm'''. It is important to ensure your construction is compatible with either of the two distinct stage mounting platforms available in the 20.309 lab (either Newport or Thorlabs model). If you find it inconvenient to measure this, there is a Handy Scope Height Thingama-jig floating around the lab. Ask your instructor(s). Also, note that the stages are very expensive; always lift them from the bottom.<br />
* Verify the focal length of the lenses you selected. If you find an optic in the wrong box: identify the optic and replace it in the correct box or label the box correctly. (Ask an instructor if you can't find the right box. There are many boxes near the wire spools behind you as you stand at the wet bench.)<br />
* Check all your lenses for cleanliness before you use them. You'll save yourself some troubleshooting time and effort down the road!<br />
* Make sure all your components are "leveled" (horizontal, not slanted).<br />
* Use tube rings (and never an SM1T2, SM1V01, or SM1V05) to mount optics in lens tubes.<br />
* Use adjustable mounting components in front of the CCD camera so you can optimize and fine-tune the camera positing with respect to the imaging lens ''L2''. Beware: never use an SM1T2 coupler without a locking ring &mdash; they are very difficult to remove if they are tightened against a lens tube or tube ring. Also put a quick-connect in your design such that the camera CCD will end up 200 mm from the back focal plane of the objective. Remember that the CCD is recessed inside the opening of the camera.<br />
[[Image:20.309 130816 CCD QuickConnect.png|center|thumb|400px|Adjustable Thorlabs SM1V05 and SM1T2 connectors precede the quick-connect union to the CCD camera.]]<br />
* Restrict to 3 struts only the connection between the cage cube and the last silver mirror before the CCD camera, so you can easily take in and out the barrier filter ''BF'' that will later aid fluorescence-mode microscopy.<br />
[[Image:20.309 130816 BarrierFilterSpace.png|center|thumb|250px| Insertion and removal of optical components is facilitated by a three-strut-only link. ]]<br />
<br />
* The Nikon objective lenses are designed to be paired with a 200 mm tube lens.<br />
* Assume that the objectives behave as ideal plano-convex lenses.<br />
* Fine focusing will be achieved by adjusting the height of the sample stage.<br />
* ''Tip:'' Throughout the optical microscopy lab, start the alignment with a 10× objective but progress to 40× and 100×.<br />
* You can use either a red or a blue LED illuminator for bright-field transmitted light imaging. <br />
** Each group will receive their own LED. Please ask an instructor if you cannot find one.<br />
{{Template:Safety Warning|message=Double check your wiring before powering the LED. The LED can be damaged by excessive current. Limit the driving current to 0.5 A to protect the LED.}}<br />
<br />
==Magnification measurement==<br />
[[Image:20.309_130813_BrightFieldExampleImages.png|right|thumb|Example images included by past students in their Week 1 report: (top) Air Force target, (center) Silica spheres and dust, (bottom) Ronchi Ruling]]<br />
Measuring the magnification of your microscope is a good way to verify that your instrument is functioning well. You should measure the magnification of any microscope you plan to use for making quantitative measurements of size. Use the measured value in your calculations, not the number printed on the objective. Consider the uncertainty in your measurement.<br />
<br />
# Use MATLAB's Image Acquisition Tool to view a live display and record images<br />
#* Launch Matlab and type <tt>imaqtool</tt>. An Image Acquisition Tool window should fill the screen.<br />
#* Select the "Mono12" mode of the "Manta_G-032B (gentl-1)" camera in the Hardware Browser pane.<br />
#* Once it behaves, this setting will configure the camera to produce 12-bit, monochrome images. In this mode, the intensity of each pixel in the image will be represented by 12 binary digits, allowing a range of values from 0-4095.<br />
#* In the Acquisition Parameters pane, select the "Device Properties" tab and set "Acquisition Frame Rate Abs" to 20. This will cause the camera to take 20 complete images per second.<br />
#* Click the "Start Preview" button. The live image from the camera should appear in the Preview pane.<br />
#* If this does not produce a live image, close the window, issue the command imaqreset in the Matlab workspace. Then issue the command imaqtool again, choose the "gige-1" driver and start it as above. Regardless of whether it works, now close the window, issue the imaqreset command again, then the imaqtool command, and continue by choosing the "gentl-1" option. The drivers are, it goes without saying, a little flaky. It is best to avoid the "gige-1" option for long-running because it is more prone to hanging up than the "gentl-1" option.<br />
#* Use the "Exposure Auto" and "Exposure Time Abs" settings in the "Device Properties" tab to produce a good image. Setting "Exposure Auto" to "Once" will cause the camera to run its automatic exposure algorithm one time. This usually results in an exposure that's in the ballpark. But the automatic exposure usually does a poor job on microscopic images. Make the exposure better by changing the value in "Exposure Time Abs". The value sets the exposure time for each frame in microseconds.<br />
# Ensure that the camera's field of view is approximately centered in the objective's field of view. <br />
#* The objective has a larger FOV than the camera. Use the adjustment knobs on mirror M1 to traverse the objective's FOV horizontally and vertically. The FOV is approximately circular. Find a spot near the middle.<br />
# Measure the magnification of your microscope using the 10x, 40x, and 100x objectives.<br />
#* Start with the 10x objective and an Air Force imaging target.<br />
#** There are two styles of Air Force imaging target available in the lab. Both have sets of precisely spaced vertical and horizontal, high-contrast black/white line pairs. One version of the target indicates the size of the line pairs with a number conveniently printed near the set of lines. The number indicates how many black/white line pairs per millimeter. The other style of Air Force imaging target uses an annoying group/element designation that is explained on [[http://en.wikipedia.org/wiki/1951_USAF_resolution_test_chart this Wikipedia page]]. <br />
#** Make sure that the side of the target with the pattern on it faces the objective. Imaging through the thick glass causes distortion and many other troubles.<br />
#* Record an image of appropriately sized lines on the Air Force imaging target (with 10x and 40x objectives) and the Ronchi ruling (with the 100x objective).<br />
#** Why is it not judicious to image the Ronchi ruling with the 10x objective?<br />
#* Set "Frames per trigger" setting in the "General" tab of the Acquisition Parameters pane to 1. This setting controls how many images MATLAB will record each time you press the "Start Acquisition" button. <br />
#* Press the "Start Acquisition" in the Preview Pane. (The live preview will stop.)<br />
#* Press "Export Data..." In the dialog that comes up, select "MATLAB Workspace" in the "Data Destination" popup and type in a variable name, such as <tt>AirForce14lp10x</tt>. Data from the image you acquired will be available in the Matlab workspace.<br />
#* Switch to the MATLAB command window and type <tt>whos AirForce14lp10x</tt>. The image is represented as a 492x656 matrix of 16-bit integers.<br />
#* To display the image, use the <tt>imshow</tt> command. <br />
#** When the 12-bit numbers from the camera get transferred to the computer, they are converted to 16-bit numbers. 16-bit numbers can represent a range of values from 0-65535. This leaves a considerable portion of the number range unoccupied. Because of this, if you type <tt>imshow( AirForce14lp10x )</tt>, you will see an image that looks almost completely black. Adjust the image to fill the full range by typing <tt>imshow( 16.0037 * AirForce14lp10x )</tt>. 16.0037 equals 65535/4095. This factor maps values in the range 0-4095 to 0-65535.<br />
#* An even better way to work with images in MATLAB is to convert them to [[http://en.wikipedia.org/wiki/Double-precision_floating-point_format double precision floating point format]] straightaway. Double precision floating point numbers can represent an extremely wide range of values with high precision. Matlab includes a function for converting images, <tt>im2double</tt>. Type <tt>AirForce14lp10x = im2double( 16.0037 * AirForce14lp10x );</tt> to make the conversion and then use <tt>imshow</tt> to see the result. After the conversion to double, the range of intensities is mapped to 0-1, with 1 being full intensity and 0 completely dark.<br />
#* Use the data cursor to measure magnification<br />
#** When choosing the size of lines to image, consider the factors that influence the uncertainty of your measurement.<br />
#* Save your image as a .mat for later use in MATLAB (<tt>save AirForce14lp10x</tt>) or as a PNG image for use in your report or other programs. if you converted the image to double, the command might look something like: <tt>imwrite( im2uint16( AirForce14lp10x ), 'AirForce14lp10x.png', 'png' );</tt>.<br />
# Repeat the magnification measurement for the 40x and 100x objectives. <br />
#* With the 100x objective, you may want to substitute the Air Force target with a Ronchi Ruling, a grating with 600 line pairs per millimeter.<br />
# Calculate the FOV of the microscope using all three objectives.<br />
<br />
==Particle size measurement==<br />
[[Image:20.309_130813_BF_3p2umbeads_40x.png|right|thumb|Example image of 3.2 μm beads using the instructor microscope. Submit picture to replace this!]]<br />
<br />
Now that you know the magnification of your instrument, use it to measure the size of some microscopic objects as imaged with the 40x objective lens only. Slides with 7.2 μm, 3.2 μm and 1 μm silica microspheres are available in the lab.<br />
<br />
# Image 7.2 μm, 3.2 μm and 1 μm silica microspheres as described in the magnification measurement procedure (40x objective only). <br />
# Measure and report the average size and uncertainty of the spheres in each sample. How many spheres should you measure?<br />
<br />
==Microscope storage==<br />
<br />
During the microscopy lab, approximately seven thousand optical components will be taken from stock, assembled into microscopes, and properly returned to their assigned places. Please observe the following:<br />
* Store your microscope in one of the cubby holes in 16-336 (not in the lab). If you use one of the high shelves, get somebody to help you lift.<br />
* Keep all of the boxes for the optics you use with your instrument to simplify putting things away. <br />
* Take a blue bin to store loose items (such as lens boxes) in.<br />
* Stages, CCD cameras, neutral density filters and barrier filters stay at the lab station. Do not store these with your microscope.<br />
* Return objective lenses to the drawer when you are not using them. (Do not store them with your microscope.)<br />
* The stages are very expensive. Always lift from the bottom.<br />
* If you break something (or discover something pre-broken for you), do not return it to the component stock. Give all broken items to an instructor. You will not be penalized for breaking something, but not reporting may be looked upon less kindly.<br />
<br />
==Report outline==<br />
<br />
Find and follow all guidelines on the [[Microscopy report outline]] wiki page. <br />
<br />
{{:Optical Microscopy: Part 1 Report Outline}}<br />
<br />
{{:Optical microscopy lab wiki pages}}<br />
<br />
==References==<br />
<references /><br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/DNA_Melting_Report_RequirementsDNA Melting Report Requirements2015-11-24T20:24:44Z<p>Steven Nagle: /* Part 2 report outline */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
<br />
__NOTOC__<br />
* Follow the [[20.309:Lab Report Guidelines|lab report general guidelines]].<br />
* Provide a thorough and accurate discussion of error sources, measurement uncertainty, and confidence in your results. An outstanding error discussion is an essential element of a top-notch report.<br />
* One member of your group should submit a single PDF file to Stellar in advance of the deadline. The filename should consist of the last names of all group members, CamelCased, in alphabetical order, with a .pdf extension. Example: <code>CrickFranklinWatson.pdf</code>.<br />
<br />
==Part 2 report outline==<br />
# Haiku:<br />
#* Compose an entertaining, exhilarating, thought-provoking, or melancholy Haiku on the subject of DNA melting.<br />
# Abstract: <br />
#* In one paragraph containing six or fewer sentences, summarize the investigation you undertook and key results.<br />
# Introduction and Purpose:<br />
#* Provide a succinct introduction to the project, including the purpose of the experiment, relevant background material and/or links to such information.<br />
#* Summarize the ways in which this part of the lab differs from Part 1.<br />
#* Keep the length to one or two short paragraphs, no more than 1/3 of a page.<br />
# Apparatus:<br />
#* Document your instrument design.<br />
#** Describe your apparatus with sufficient detail for another person to replicate your work. Assume the reader is familiar with the concepts of 20.309 and has access to course materials.<br />
#** Detail your electronic and optical subsystems. Include component values, gain values, cutoff frequencies, lens focal lengths, and relevant distances.<br />
#** It is not necessary to document construction details, unless you built an instrument that was significantly different than the lab manual suggested.<br />
#** Refer to schematics and diagrams in the lab manual instead of copying them into your report. Use reference designators (such as R7, C2) to refer to call out particular components in schematic diagrams.<br />
#* Why not include a nice snapshot or two of the instrument? So lovely.<br />
# Procedure:<br />
#* Document the procedure used to gather your data. <br />
#** Refer to procedures in the lab manual. Describe any changes you made.<br />
#** Report instrument settings for each trial, including control software parameters.<br />
# Data:<br />
#* Plot all of your raw data, fluorescence vs. block temperature, on the smallest number of axes that clearly conveys the dataset. Include only data generated by your own group. <br />
#** Data from the many sample runs overlaps, which makes presenting so much data on a small number of axes a real challenge. <br />
#** Devise a combination of line colors, line thicknesses, and marker symbols that produces clear plot. If two sample types have a great deal of overlap, there may be no choice but to plot them on separate axes.<br />
#** One approach that works well for some datasets is to plot a subsampled version of each trial using discrete markers. Vary the color and form to differentiate between sample types and individual trials.<br />
#* Report your signal to noise results.<br />
# Analysis:<br />
#* Use bullet points to explain your data analysis methodology.<br />
#* Document the regression model you used to analyze your data<br />
#** See [[DNA Melting: Model function and parameter estimation by nonlinear regression]]<br />
#** Explain the model parameters using bullet points or in a table.<br />
#* Plot <math>V_{f,measured}</math> and <math>V_{f,model}</math> versus <math>T_{block}</math> for a typical run of each samples type. Use the smallest number of axes that clearly conveys the data. <br />
#* For a typical curve, plot residuals versus time, temperature, and fluorescence, ([http://measurebiology.org/wiki/File:Residual_plot_for_DNA_data.png example plot]).<br />
#* Provide a table of the best-fit model parameters and confidence intervals for each experimental run. Also include the estimated melting temperature for each run.<br />
#* For at least one experimental trial, plot <math>\text{DnaFraction}_{inverse-model}</math> versus <math>T_{sample}</math> ([http://measurebiology.org/wiki/File:Inverse_cuvrve.png example plot]). On the same set of axes plot DnaFraction versus <math>T_{sample}</math> using the best-fit values of &Delta;H and &Delta;S. Finally, plot simulated dsDNA fraction vs. temperature using data from DINAmelt or another melting curve simulator.<br />
# Results:<br />
#* Identify your unknown sample (or state that your investigation did not provide a conclusive answer).<br />
#* Quantify the confidence you have in your result.<br />
# Discussion:<br />
#* Discuss the validity of assumptions in the regression model.<br />
#* Discuss any atypical results or data you rejected.<br />
#* Compare your data to results from other groups and/or instructor data.<br />
#* Give a bullet point summary of problems you encountered in the lab during part 2 and changes that you made to your instrument and methodology to address those issues.<br />
#* Discuss significant error sources. <br />
#** Consider the entire system: the oligos, dye, the experimental method, and analysis methodology, and any other relevant factors.<br />
#** Indicate whether each source likely caused a systematic or random distortion in the data. <br />
#** Present error sources, error type and their resultant uncertainty on your data and results in a table, if you like.<br />
#* Discuss additional unimplemented changes that might improve your instrument or analysis.<br />
<br />
==Lab manual sections==<br />
<br />
*[[Lab Manual:Measuring DNA Melting Curves]]<br />
*[[DNA Melting: Simulating DNA Melting - Basics]]<br />
*[[DNA Melting Part 1: Measuring Temperature and Fluorescence]]<br />
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]<br />
*[[DNA Melting Report Requirements]]<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/File:Inverse_cuvrve.pngFile:Inverse cuvrve.png2015-11-24T20:07:46Z<p>Steven Nagle: </p>
<hr />
<div></div>Steven Naglehttp://measurebiology.org/wiki/DNA_Melting_Report_RequirementsDNA Melting Report Requirements2015-11-24T20:06:44Z<p>Steven Nagle: </p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
<br />
__NOTOC__<br />
* Follow the [[20.309:Lab Report Guidelines|lab report general guidelines]].<br />
* Provide a thorough and accurate discussion of error sources, measurement uncertainty, and confidence in your results. An outstanding error discussion is an essential element of a top-notch report.<br />
* One member of your group should submit a single PDF file to Stellar in advance of the deadline. The filename should consist of the last names of all group members, CamelCased, in alphabetical order, with a .pdf extension. Example: <code>CrickFranklinWatson.pdf</code>.<br />
<br />
==Part 2 report outline==<br />
# Haiku:<br />
#* Compose an entertaining, exhilarating, thought-provoking, or melancholy Haiku on the subject of DNA melting.<br />
# Abstract: <br />
#* In one paragraph containing six or fewer sentences, summarize the investigation you undertook and key results.<br />
# Introduction and Purpose:<br />
#* Provide a succinct introduction to the project, including the purpose of the experiment, relevant background material and/or links to such information.<br />
#* Summarize the ways in which this part of the lab differs from Part 1.<br />
#* Keep the length to one or two short paragraphs, no more than 1/3 of a page.<br />
# Apparatus:<br />
#* Document your instrument design.<br />
#** Describe your apparatus with sufficient detail for another person to replicate your work. Assume the reader is familiar with the concepts of 20.309 and has access to course materials.<br />
#** Detail your electronic and optical subsystems. Include component values, gain values, cutoff frequencies, lens focal lengths, and relevant distances.<br />
#** It is not necessary to document construction details, unless you built an instrument that was significantly different than the lab manual suggested.<br />
#** Refer to schematics and diagrams in the lab manual instead of copying them into your report. Use reference designators (such as R7, C2) to refer to call out particular components in schematic diagrams.<br />
#* Why not include a nice snapshot or two of the instrument? So lovely.<br />
# Procedure:<br />
#* Document the procedure used to gather your data. <br />
#** Refer to procedures in the lab manual. Describe any changes you made.<br />
#** Report instrument settings for each trial, including control software parameters.<br />
# Data:<br />
#* Plot all of your raw data, fluorescence vs. block temperature, on the smallest number of axes that clearly conveys the dataset. Include only data generated by your own group. <br />
#** Data from the many sample runs overlaps, which makes presenting so much data on a small number of axes a real challenge. <br />
#** Devise a combination of line colors, line thicknesses, and marker symbols that produces clear plot. If two sample types have a great deal of overlap, there may be no choice but to plot them on separate axes.<br />
#** One approach that works well for some datasets is to plot a subsampled version of each trial using discrete markers. Vary the color and form to differentiate between sample types and individual trials.<br />
#* Report your signal to noise results.<br />
# Analysis:<br />
#* Use bullet points to explain your data analysis methodology.<br />
#* Document the regression model you used to analyze your data<br />
#** See [[DNA Melting: Model function and parameter estimation by nonlinear regression]]<br />
#** Explain the model parameters using bullet points or in a table.<br />
#* Plot <math>V_{f,measured}</math> and <math>V_{f,model}</math> versus <math>T_{block}</math> for a typical run of each samples type. Use the smallest number of axes that clearly conveys the data. <br />
#* For a typical curve, plot residuals versus time, temperature, and fluorescence, ([http://measurebiology.org/wiki/index.php?title=File:Residual_plot_for_DNA_data.png example plot]).<br />
#* Provide a table of the best-fit model parameters and confidence intervals for each experimental run. Also include the estimated melting temperature for each run.<br />
#* For at least one experimental trial, plot <math>\text{DnaFraction}_{inverse-model}</math> versus <math>T_{sample}</math> ([http://measurebiology.org/wiki/index.php?title=File:Inverse_cuvrve.png example plot]). On the same set of axes plot DnaFraction versus <math>T_{sample}</math> using the best-fit values of &Delta;H and &Delta;S. Finally, plot simulated dsDNA fraction vs. temperature using data from DINAmelt or another melting curve simulator.<br />
# Results:<br />
#* Identify your unknown sample (or state that your investigation did not provide a conclusive answer).<br />
#* Quantify the confidence you have in your result.<br />
# Discussion:<br />
#* Discuss the validity of assumptions in the regression model.<br />
#* Discuss any atypical results or data you rejected.<br />
#* Compare your data to results from other groups and/or instructor data.<br />
#* Give a bullet point summary of problems you encountered in the lab during part 2 and changes that you made to your instrument and methodology to address those issues.<br />
#* Discuss significant error sources. <br />
#** Consider the entire system: the oligos, dye, the experimental method, and analysis methodology, and any other relevant factors.<br />
#** Indicate whether each source likely caused a systematic or random distortion in the data. <br />
#** Present error sources, error type and their resultant uncertainty on your data and results in a table, if you like.<br />
#* Discuss additional unimplemented changes that might improve your instrument or analysis.<br />
<br />
==Lab manual sections==<br />
<br />
*[[Lab Manual:Measuring DNA Melting Curves]]<br />
*[[DNA Melting: Simulating DNA Melting - Basics]]<br />
*[[DNA Melting Part 1: Measuring Temperature and Fluorescence]]<br />
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]<br />
*[[DNA Melting Report Requirements]]<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/DNA_Melting_Part_1:_Measuring_Temperature_and_FluorescenceDNA Melting Part 1: Measuring Temperature and Fluorescence2015-11-24T18:21:30Z<p>Steven Nagle: </p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
<br />
[[Image:DNA Melting Block Diagram.jpg|frame|DNA melting apparatus block diagram showing the five functional groups of the instrument.]]<br />
<br />
<br />
''In theory, there is no difference between theory and practice. But, in practice, there is.'' <br />
<br />
''&mdash;[http://en.wikiquote.org/wiki/Jan_L._A._van_de_Snepscheut Jan L. A. van de Snepscheut]/[http://en.wikiquote.org/wiki/Yogi_Berra Yogi Berra]/and or [http://c2.com/cgi/wiki?DifferenceBetweenTheoryAndPractice Chuck Reid]'' <br />
<br />
You will construct a basic DNA melting instrument consisting of the five functional groups shown in the block diagram:<br />
* excitation<br />
* heating<br />
* fluorescent detection<br />
* temperature measurement<br />
* data acquisition and control<br />
<br />
A metal block will hold a sample vial in the excitation light path and transfer heat to the sample. The sample contains a combination of DNA, LCGreen Plus and a salt. LCGreen Plus fluoresces when it is bound to double stranded DNA (dsDNA) by a factor of up to 1000x compared to when it is bound to single stranded DNA (ssDNA). Detection of this fluorescence provides a relative measure of the fraction of dsDNA to total DNA, which can be compared to predictions from a thermodynamic model.<br />
<br />
The dye is excited by blue light, which will be delivered by a low power LED. The dye emits green light. A photodiode will be placed at 90&deg; to the LED source to detect the green light emitted by LCGreen Plus. Because LEDs have a fairly broad spectrum, first a moderately narrow band-pass filter will be used to limit the excitation wavelength to between 450 and 490 nm. Then a complimentary long-pass emission filter will be placed between the sample and detector to pass only wavelengths longer than 515 nm. This filter combination, when used with this dye system, eliminates any background signal due to the excitation light.<br />
<br />
The sample block will be heated by a TEC (thermoelectric cooler) and its temperature measured by an embedded RTD (resistance temperature detector). Also, since photodiodes produce a very small amount of current, a high gain amplifier will convert the photodiode current into a measurable voltage. The DAQ (data acquisition card) and PC will digitize the amplified photodiode and RTD signals. A Matlab program is provided to record these fluorescence and temperature signals over time and save the data to a file. The file can then be loaded into Matlab for analysis, or if you prefer, Python or equivalent where the signals will be converted into dsDNA concentration and sample temperature. Finally, the dsDNA sample melting temperature will be estimated from a plot of df/dT vs T (''f'' represents the fraction of total DNA that is in double-stranded form) and from a comparison to the thermodynamic model.<br />
<br />
The functional groups in the block diagram are one of two fundamental frameworks used to describe an instrument. The other sub-divides the instrument into categories that often mirror historical areas of engineering. The former is focused on what will be done, including the overall scientific goals and the physical processes to be measured. The latter is focused on how it will be done, e.g. using optics, electronics or perhaps a practical knowledge of thermal management. A good engineer will constantly be thinking of the instrument in both frameworks. <br />
<br />
==Mechanical and thermal components==<br />
Mechanical support for the heating block, sample and optics will be built on a portable optical breadboard from [http://www.thorlabs.com ThorLabs]. Most of the components in the lab are from this supplier because the selection is large, the quality is quite good and the price reasonable. Although you are free to modify the design as the lab progresses, begin a build from the standard design shown above and demonstrated by the example in the lab. Gather the following Thorlabs components but do not start to assemble.<br />
<br />
* 1 MB1224 optical breadboard<br />
* 2 CL5 clamps (or similar)<br />
* 2 1/4-20 5/8" screws<br />
* 4 1/4-20 1/4" screws<br />
<br />
The thermal subsystem includes all components to control heat flow into and out of the sample and monitor its temperature. Heating is accomplished using thermal electric coolers (TECs) in reverse. The hot side is placed in direct contact with the heating block, which is in direct contact with the sample vial. The TECs will be powered by manually controlling the Diablotek power supply. To avoid contamination of the sample or perturbation of its temperature, the temperature of a resistive temperature detector (RTD) embedded in the heating block will be monitored as a proxy for the sample temperature. In this part of the lab, air convection will cool down the sample when the Diablotek is switched off. The heat sink will help and the fan may be connected to a triple-output power supply to help, but these are included primarily for use in Part 2.<br />
<br />
===Parts===<br />
Gather the following components from the bins on the Center Cabinets, the DNA Melting drawer at the wet bench or on the East Cabinets:<br />
<br />
[[Image:CenterCabinets.jpg|right|250px|thumb|'''Center Cabinets''' The bins for most parts that you need will be set out on top of these cabinets, roughly in the center of the lab.]]<br />
[[Image:WetBench.jpg|right|250px|thumb|'''Wet Bench''' Supplies for our wet bench can be found in the three drawers at the left of the bench, the drawer left of the sink and on the wall behind and to the left as you face the wet bench. The drawer at the right of the wet bench contains various supplies for the DNA Melting instrument.]]<br />
[[Image:EastCabinets.jpg|right|250px|thumb|'''East Cabinets''' The center shelves of the East Cabinets contain the electronic breadboards, breadboard jumper wire, TEC connectors and TEC power supplies. Often these supplies are laid out on top of these cabinets.]]<br />
<br />
* These components are already assembled for you: A metal support bracket, having a fan, heatsink, 2 TEC heaters/coolers, and a heating block attached to it<br />
* 3 wire nuts<br />
* 1 Diablotek 250 W power supply (at your station or on a shelf above your station)<br />
* 1 red/black TEC extension wire assembly (in a box on the ''''Center Cabinets'''')<br />
<br />
===Assembly instructions===<br />
<br />
[[Image:TEC_picture.jpg|150 px|thumb|right|TEC showing hot side up when current flows into the red lead; i.e., when a positive voltage is applied between the red and black leads.]]<br />
<br />
# The bracket, TECs, fan and heatsink should already be assembled for you.<br />
#* The TECS are sandwiched between the heating block (on top) and the heat sink (underneath).<br />
# Verify that the colors of the leads match. If not, flip one TEC with reference to the picture at right.<br />
# Connect the black lead of the top TEC to the red lead of the bottom TEC using a wire nut.<br />
# Connect the TEC to the black/white (and black/yellow) extension wire assembly using wire nuts to join the white wire to the red lead of the top TEC and the black wire to the black lead on the bottom TEC, respectively.<br />
# Join the white connector on the assembly to the '''black''' 2x2 connector of the Diabloteck.<br />
#* Use only the '''black''' 2x2 connector, not the white one.<br />
# Test the TECs by touching the base of your heating block and turning the Diablotek on.<br />
#* The base should start to feel warm within 10-15 seconds.<br />
#* If the Diablotek immediately shuts off (the fan will stop spinning) then there is probably a short. Check all connections.<br />
# Once the TEC assembly wiring is checked, try the heating test again.<br />
# If it fails again, find an Instructor.<br />
<br />
==Optical subsystem==<br />
<br />
A good optical subsystem design will be compact, stable and simple. It includes the optical components of the excitation and fluorescence detection functional groups from the block diagram. A good design will efficiently illuminate the sample and collect the maximum possible amount of emitted fluorescent light. There are a variety of ways to construct the apparatus. Use the Instructor instrument(s), and the instructions below, as a guide. The set of instructions can help if you're not clear on how to replicate the example system in the lab. In addition to the following parts, you might want to add a few lenses to collimate your LED onto your sample, and to focus the light coming from your sample onto your photodiode. <br />
<br />
* 2 PH2 post holders<br />
* 2 BA1 post holder bases<br />
* 2 1/4-20 1/4" screws<br />
* 2 1/4-20 5/8" screws<br />
* 2 1/4" washers<br />
* 4 CP02 cage plates<br />
* 2 8-32 1/2" setscrews<br />
* 2 TR2 posts<br />
* 6 ER3 cage rails<br />
* 12 4-40 1/4" setscrews (or similar length)<br />
* 4 SML05 lens tubes<br />
* 4 SM1LTRR retaining rings<br />
* 1 blue LED PCB<br />
* 1 D470/40 excitation filter<br />
* 1 E515LPv2 emission filter<br />
* 1 SM1A6 adapter between SM1 and SM05 threads<br />
* 1 SM05PD1A mounted photodiode<br />
<br />
===Assembly instructions===<br />
Use an Instructor instrument or the Solid Works model above as guide to assemble your optical subassembly. Note also that these instructions are also a guid. If you wish to build your own personal design, please do.<br />
<br />
# Attach each PH2 to a BA1 using 1/4" screws.<br />
# Attach two of the CP02 cage plates to two TR2 posts using an 8-32 setscrew.<br />
# Insert the TR2/CP02 assembly into the PH2/BA1 assemblies and lock in place with the TS25H setscrews.<br />
# Insert three ER4 cage rails into each CP02 above and lock in place with SS4S025 set screws or similar.<br />
#* Leave the blank hole on the bottom of the assembly.<br />
# Attach one of the remaining CP02 cage plates on the free end of each assembly above and lock in place.<br />
# Attach each of the above assemblies to the MB1224 using a washer and 1/4-20 screw less than 1" in length.<br />
# Arrange on the MB1224 and adjust CP02 heights as desired.<br />
# Mount the LED PCB in an SML05 lens tube and fix with a retaining ring.<br />
# Mount the excitation and emission filters in each of two lens tubes and fix with retaining ring.<br />
#* The arrow label on the thickness of the filter points ''toward the light source''.<br />
# Thread the SM1A6 adapter into a lens tube and fix with a retaining ring.<br />
# Thread the photodiode into the SM1A6 adapter.<br />
#* Teflon pipe thread tape can be used to hold the photodiode in the desired position.<br />
# Mount each lens tube in the appropriate CP02 cage plates.<br />
# Arrange each the resultant assemblies on the breadboard to most fully illuminate the sample vial and to gather the most of the resultant light on the photodiode.<br />
<br />
==Electronics subsystem==<br />
<br />
The electrical subsystem includes the LED with a current-limiting resistor, the photodiode, the transimpedance amplifier and a temperature detection circuit. The remainder of this section provides an example of each of these systems and instructions to build and test each one. Nevertheless, alternate ideas are welcome. Feel free to discuss with the Instructors.<br />
<br />
===Parts===<br />
<br />
Gather the following components from the electronics drawer to the right of the optics drawers, from the counter top opposite the wet bench and from the cable organizer on the East wall to the right of the white board.<br />
* 1 electronic breadboard<br />
* 1 jump wire kit<br />
* 2 red 3' banana cable or similar<br />
* 1 black 3' banana cable or similar<br />
* individual wire, as-needed from the scrap wire box or the wire spools opposite the wet bench<br />
* 1 62 &Omega; resistor from the center counter top on the South wall<br />
* 1 15 k&Omega; resistor<br />
* other resistors, as-needed for the amplifier<br />
* 1 blue LED from the DNA Melting drawer at the far right of the wet bench<br />
* 2 LF411 op-amps from the electronics drawer<br />
* 4 capacitors between 0.1 and 22 uF from either the electronics drawer or the counter top where the screws are stored.<br />
<br />
===Assembly instructions===<br />
<br />
====LED circuit====<br />
The led circuit consists of a 62 &Omega; resistor in series with a blue, jumbo LED. The circuit will be powered by the 5 volt fixed output of the lab power supply. The 62 &Omega; resistor prevents excessive current from flowing through the LED. Excessive current will let the smoke out of the LED.<br />
<br />
# Build the LED circuit on your electronic breadboard.<br />
#* Don't forget to connect the resistor. Directly connecting the LED to 5 V will blow out the LED.<br />
# Disable the output of the lab power supply.<br />
# Connect the LED circuit to the fixed 5 V output.<br />
<br />
====Temperature detection circuit====<br />
A platinum RTD is embedded inside the heating block. The resistance of the RTD varies with temperature according to the equation ''R<sub>RTD</sub>''=1000 &Omega; + 3.85 ''&theta;'', where ''&theta;'' is the temperature of the RTD in degrees Celsius. The easiest way to make a temperature measurement is to connect the RTD in series with a fixed resistor and a power supply. The output voltage of the resulting divider circuit varies as a function of temperature. <br />
<br />
The maximum current of the RTD is 1 mA. You will use a 15 V supply to the circuit, so the resistor must be at least 14 k&Omega;. Use a 15 k&Omega; resistor.<br />
<br />
# '''Find an equation for ''V<sub>out</sub>'' as a function of temperature. What voltage do you expect given an input voltage of 15 V with the RTD at room temperature.'''<br />
# Turn off the output of the triple-output power supply.<br />
# Connect the RTD to the electronic breadboard.<br />
# '''Measure the resistance of the 15 k&Omega; resistor with a DMM and record the value in your lab report.'''<br />
# Connect the 15k&Omega; resistor in series with the RTD.<br />
# Set the power supply for '''SERIES''' operation.<br />
# Set the output voltage to 15 V on the supply and make note of its exact value using the DMM.<br />
# Set the current limit to 0.1 A.<br />
# Connect the positive terminal of '''CH1''' to a banana terminal on the breadboard for +15 V and wire it to the top red bus strip for +15 V supply.<br />
# Connect the negative terminal of '''CH2''' to a banana terminal on the breadboard for -15 V.<br />
# Connect the positive terminal of '''CH2''' to the black terminal on the breadboard for a ground reference and wire it to the top blue bus strip.<br />
#* Use three jump wires to minimize the parasitic voltage drop due to the breadboard tie points.<br />
# Connect the +15 V bus strip to the second vertical, red bus strip and wire this bus strip to the top of your 15 k&Omega; resistor.<br />
# Turn on the output of the power supply and measure the voltage across the RTD using the DMM.<br />
# '''What is the measured voltage of the RTD at room temperature and how does it compare to your predictions above?'''<br />
# '''What is the temperature, based on your measured voltage and how does it compare with the clock on the wall (or some better measure)?'''<br />
# '''How is the temperature calculation affected by a 1% change in the 15 V input voltage?'''<br />
<br />
====Photodiode amplifier circuit====<br />
<br />
The current produced by the photodiode detector is a few tens of nanoamperes. This very small current must be amplified to a level where it can be easily measured. The data acquisition system you will use can measure voltages between + and -10 V. Use the two-stage op amp circuit shown below to convert the photodiode current to a voltage.<br />
<br />
<center>[[Image:Part1_TransimpedanceAmplifier.jpg|700 px|thumb|center|Multi-stage photodiode amplification circuit]]</center><br />
<br />
You will have to decide how to distribute the gain between the two op amp stages in the circuit. It is best to put as much gain in early stages of a multi-stage amplifier as possible. However, when the gain of a single stage becomes very high, nonidealities of the op amp become bothersome. The first stage of the amplifier will behave reasonably well as long as the gain is less than about 10<sup>6</sup> &Omega;.<br />
<br />
# '''Analyze the photodiode amplifier circuit. Choose resistor values to achieve an overall gain that will yield an output of about 5 V when the input is 500 nA.'''<br />
# Construct the circuit on your breadboard.<br />
#* Disable the power supply before you build.<br />
#* Do not yet connect the photodiode.<br />
#* Orient the LF411 op-amps so that pin 1, which is marked with a dot on the package, is at the upper left.<br />
# Connect the +/- 15 V bus strips to power the op-amps power pins.<br />
# Connect the vertical blue bus strips to the horizontal blue bus strip.<br />
<br />
=====Test connectivity and basic operation=====<br />
<br />
# Verify that all your component connections are correct and match the design.<br />
# Check that wires and components are properly seated in the breadboard and not suspiciously loose.<br />
#* You can use the ohm-meter function of the digital multi-meter to verify continuity.<br />
# Turn on the power supply and verify that +15V appears on pin 7 and -15V on pin 4.<br />
# '''Disconnect the photodiode connection to pin 2 (the <math>v^-</math> input to the op-amp) and measure the output voltages of IC1 and IC2.'''<br />
#* With the photodiode disconnected, the current signal is zero. If you see a significant offset voltage (from 50 to 100 mV) at IC1 pin 6, then you may wish to add (or adjust the value of) <math>R_6</math>.<br />
#* Refer to [http://measure.mit.edu/~20.309/wiki/index.php?title=Real_electronics#Real_op-amps Real Electronics, real op-amps] or ask an instructor to understand and address this non-ideal behavior.<br />
<br />
==Data acquisition hardware subsystem==<br />
<br />
{{:20.309:DAQ System}}<br />
<br />
====Summary of DAQ inputs/outputs====<br />
<br />
A special connector has been prepared for you, connected as shown below. The connections and cable wire colors are also summarized [http://dl.dropbox.com/u/12957607/DAQ_Cable_Assembly_for_DNA_melting_lab.pdf in a memo].<br />
<br />
{| class="wikitable"<br />
|-<br />
! Signal Name<br />
! Signal Location<br />
! Ground Location<br />
! Pin wire color<br />
|-<br />
! DAQ Inputs <br />
|-<br />
! RTD<br />
| AI0+<br />
| AI0-<br />
| +Orange / -Black [lone pair, not with third (red) wire]<br />
|-<br />
! Photodiode <br />
| AI1+ <br />
| AI1-<br />
| +Green / -White<br />
|}<br />
<br />
<center>[[Image:DAQ_Wire_Colors.png|450 px|thumb|center|DAQ Connection Cable]]</center><br />
<br />
==DNAMelter software==<br />
<br />
[[Image:BasicDNAMelterIcon.png|75 px|left]]<br />
Use the <code>Basic DNA Melter GUI</code> program (located on the lab computer desktop) to collect data from the apparatus.<br />
<br />
At the end of an experimental run, use the "Save" button to write the date to a file. The data will be tab-delimited and can be read into Matlab with the <code>load</code> command.<br />
<br />
Documentation for the DNAMelter software is available here: [[DNA Melting: Using the Basic DNAMelter GUI]]<br />
<br />
====If you need to debug the DAQ (skip otherwise!)====<br />
* When debugging problems, it is frequently useful to ascertain whether the data acquisition system is working properly. National Instruments provides an application for this purpose called Measurement and Automation Explorer. Use Measurement and Automation Explorer to verify that the DAQ is properly connected to and communicating with the computer. The program also provides the ability to manually control all of the DAQ system's inputs and outputs.<br />
** Launch Measurement and Automation Explorer by selecting Start->All Applications->National Instruments->Measurement and Automation.<br />
** In the "Configuration" tab, select "Devices and Interfaces" and then "NI-DAQMx Devices." Select "Dev1."<br />
** If Dev 1 does not show up, the problem is that computer cannot communicate with the DAQ system. Ensure that the USB cable is connected to the DAQ system. If the cable is connected and the device still does not show up, unplug the USB cable, count to five, and plug it back in. <br />
** If the DAQ shows up under a different name, disconnect the USB cable. Delete all the "NI USB-6xxx" entries and then reconnect the USB cable. Verify that one entry reappears and that it is called "Dev1." <br />
** Select the "Test Panels" tab to manually control or read signals from the DAQ.<br />
* If you run DNAMelter and there is a data acquisition error, for example, "<code>Data acquisition cannot start!</code>", open Measurement and Automation Explorer and follow the steps above. Run DNAMelter again. If it still doesn't work, grab a beer or ask an instructor for help.<br />
* The DAQ has only one analog-to-digital converter (ADC). A multiplexer circuit sequentially samples voltages from the analog inputs configured in the control software. If any of the inputs is outside the allowable range of +/-10V, all of the inputs may behave erratically. The voltages reported may look reasonable so this can be very misleading. If none of the DAQ readings make sense, ensure that all of the analog voltages are in the allowable range and reset the DAQ by cycling the power of the DAQ (use the switch on the 6341 or disconnect the USB of the 6212).<br />
* If the DAQ gets short-circuited, it may crash. If this happens, Matlab will frequently give a deceptive error message, for example claiming that the limits on the band-pass filter do not satisfy some condition. In this case, correct the connection and cycle the DAQ power.<br />
<br />
==Part 1 measurements==<br />
<br />
Please read this entire section before beginning. Then start at Testing the instrument, with reference back to the sample sections as-needed.<br />
<br />
===Making samples===<br />
<br />
{{Template:Biohazard warning|message=LC Green in DMSO is readily absorbed through skin. Synthetic oligonucleotides may be harmful by inhalation, ingestion, or skin absorption. Wear gloves when handling samples. Wear safety goggles at all times when pipetting the LC Green/DNA samples. Do not create aerosols. The health effects of LC Green have not been thoroughly investigated. See the LC Green and synthetic oligonucleotide under <code>../EHS Guidelines/MSDS Repository</code> in the course locker for more information.}}<br />
<br />
====Sample prep steps====<br />
<br />
#Pipet 500 &mu;L of DNA plus dye solution into a glass vial. <br />
#Pipet up to 200 &mu;L of mineral oil on top of the sample to help prevent evaporation. It will help to pipette into the corners first. Be careful not to get oil on the cuvette sides far above the sample. It will run down during your experiment and cause shifts in the photodiode signal. Place the cap loosely on the vial. <br />
#*Keep the sample vertical to make sure the oil stays on top. <br />
<br />
* You can use the same sample for several heating/cooling cycles.<br />
* Only discard a sample if you lose significant volume due to evaporation or if your signal gets too low. <br />
<br />
To clean your glass vial between samples, flush with alcohol in the waste container and rinse with water at the drain. Suck out residual liquid with the vacuum and drawn Pasteur pipette to the left of the sink.<br />
<br />
====Sample disposal====<br />
<br />
{{Template:Environmental Warning|message=Discard pipette tips with DNA sample residue in the pipette tips or the ''Biohazard Sharps'' container. Do not pour synthetic oligonucleotides with LC Green down the drain. Pour your used samples into the waste container provided in the middle of the wet bench.}}<br />
<br />
===Testing the instrument===<br />
<br />
Once your apparatus is built, use samples of fluorescein (~ 30&mu;M concentration) as a stand-in for LC Green + DNA to test the instrument. Fluorescein is cheap, non-toxic and will not bleach as quickly as LC Green.<br />
<br />
====Making a simple measurement of your instrument's low frequency SNR====<br />
<br />
Measure the signal to noise ratio (SNR) of the instrument by comparing the difference between the average signal with a sample present and with water as a null reference. This process requires a vial containing 500 uL of 30 uM fluorescein and a vial containing the same amount of water. <br />
<br />
# Run the <code>Basic DNA Melter GUI</code>. <br />
# Place a vial of DI water in your instrument.<br />
# Clear the data and wait 10 seconds.<br />
# Replace the water vial with the DNA of fluorescein vial, record for another 10 seconds, then save the data. <br />
#* Be sure that all other conditions, such as temperature, are stable throughout the test. <br />
<br />
Compute the signal to noise ratio, <math>\text{SNR}=\frac{\langle V_{fluorescein} \rangle - \langle V_{water} \rangle}{\sigma_{fluorescein} }</math>, where <math>V_{fluorescein}</math> is the portion of the data recorded with a fluorescein sample, <math>V_{water}</math> is the portion of the signal corresponding to the water sample, and <math>\sigma_{fluorescein}</math> is the standard deviation of <math>V_{fluorescein}</math>.<br />
<br />
===Experiment steps===<br />
<br />
Once the instrument is confirmed to be functioning properly, follow these instructions to generate melting curve data for a 20 bp DNA and green dye solution.<br />
<br />
#Open and run the <code>Basic DNA Melter GUI</code> in Matlab and follow the instructions there.<br />
# Confirm that block temperature is near room temperature when the Diablotek supply is off.<br />
# Test a full heat/cool cycle with no DNA: Apply heat to the block by switching the Diablotek on to increase the block temperature to 95&deg;C, toggle the Diablotek power in order to hold the temperature long enough for the fluorescence curve to reach a minimum, then allow the block to cool down by switching off the Diablotek.<br />
#* Other approaches may be used. One goal would be to heat the block more slowly, possibly by toggling the current while heating.<br />
# Now prepare a sample using the stock of "beater" (possibly labeled as "junk") DNA. Don't worry, it melts just as well as your fresh samples will melt.<br />
# Place the vial in the instrument, but keep the LED off until ready to measure the melting curve.<br />
# Using the same temperature cycle protocol as in your test above, measure at least one melting curve of the beater DNA sample. Repeat as-desired to understand instrument operation.<br />
#* Be sure to save a copy in a local directory so you can plot it in your Part 1 report.<br />
<br />
{{:DNA Melting Report Requirements for Part 1}}<br />
<br />
==Lab manual sections==<br />
<br />
*[[Lab Manual:Measuring DNA Melting Curves]]<br />
*[[DNA Melting: Simulating DNA Melting - Basics]]<br />
*[[DNA Melting Part 1: Measuring Temperature and Fluorescence]]<br />
*[[DNA Melting Report Requirements for Part 1]]<br />
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]<br />
*[[DNA Melting Report Requirements for Part 2]]<br />
<br />
==References==<br />
<references/><br />
<br />
==Subset of datasheets==<br />
(Many more can be found online or on the course share)<br />
#[http://www.ni.com/pdf/manuals/371931f.pdf National Instruments USB-6212 user manual]<br />
#[http://www.ni.com/pdf/manuals/370784d.pdf National Instruments USB-6341 user manual]<br />
#[http://www.national.com/ds/LF/LF411.pdf Op-amp datasheet]<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/DNA_Melting_Part_1:_Measuring_Temperature_and_FluorescenceDNA Melting Part 1: Measuring Temperature and Fluorescence2015-11-10T19:50:00Z<p>Steven Nagle: /* Experiment steps */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
<br />
[[Image:DNA Melting Block Diagram.jpg|frame|DNA melting apparatus block diagram showing the five functional groups of the instrument.]]<br />
<br />
<br />
''In theory, there is no difference between theory and practice. But, in practice, there is.'' <br />
<br />
''&mdash;[http://en.wikiquote.org/wiki/Jan_L._A._van_de_Snepscheut Jan L. A. van de Snepscheut]/[http://en.wikiquote.org/wiki/Yogi_Berra Yogi Berra]/and or [http://c2.com/cgi/wiki?DifferenceBetweenTheoryAndPractice Chuck Reid]'' <br />
<br />
You will construct a basic DNA melting instrument consisting of the five functional groups shown in the block diagram:<br />
* excitation<br />
* heating<br />
* fluorescent detection<br />
* temperature measurement<br />
* data acquisition and control<br />
<br />
A metal block will hold a sample vial in the excitation light path and transfer heat to the sample. The sample contains a combination of DNA, SYBR Green I and a salt. Recall that SYBR Green I fluoresces when it is bound to double stranded DNA (dsDNA) by a factor of up to 1000x compared to when it is bound to single stranded DNA (ssDNA). Detection of this fluorescence provides a relative measure of the fraction of dsDNA to total DNA, which can be compared to predictions from a thermodynamic model.<br />
<br />
The dye has a peak sensitivity to blue light at 497 nm so a blue LED will be used to illuminate the sample. The dye emits green light with an emission peak at 520 nm. A photodiode will be placed at 90&deg; to the LED source to detect the green light emitted by SYBR Green I. Because LEDs have a fairly broad spectrum, first a moderately narrow band-pass filter will be used to limit the excitation wavelength to between 450 and 490 nm. Then a complimentary long-pass emission filter will be placed between the sample and detector to pass only wavelengths longer than 515 nm. This filter combination, when used with this dye system, eliminates any background signal due to the excitation light.<br />
<br />
The sample block will be heated by a TEC (thermoelectric cooler) and its temperature measured by an embedded RTD (resistance temperature detector). Also, since photodiodes produce a very small amount of current, a high gain amplifier will convert the photodiode current into a measurable voltage. The DAQ (data acquisition card) and PC will digitize the amplified photodiode and RTD signals. A Matlab program is provided to record these fluorescence and temperature signals over time and save the data to a file. The file can then be loaded into Matlab for analysis, or if you prefer, Python or equivalent where the signals will be converted into dsDNA concentration and sample temperature. Finally, the dsDNA sample melting temperature will be estimated from a plot of df/dT vs T (''f'' represents the fraction of total DNA that is in double-stranded form) and from a comparison to the thermodynamic model.<br />
<br />
The functional groups in the block diagram are one of two fundamental frameworks used to describe an instrument. The other sub-divides the instrument into categories that often mirror historical areas of engineering. The former is focused on what will be done, including the overall scientific goals and the physical processes to be measured. The latter is focused on how it will be done, e.g. using optics, electronics or perhaps a practical knowledge of thermal management. A good engineer will constantly be thinking of the instrument in both frameworks. <br />
<br />
==Mechanical and thermal components==<br />
Mechanical support for the heating block, sample and optics will be built on a portable optical breadboard from [http://www.thorlabs.com ThorLabs]. Most of the components in the lab are from this supplier because the selection is large, the quality is quite good and the price reasonable. Although you are free to modify the design as the lab progresses, begin a build from the standard design shown above and demonstrated by the example in the lab. Gather the following Thorlabs components but do not start to assemble.<br />
<br />
* 1 MB1224 optical breadboard<br />
* 2 CL5 clamps (or similar)<br />
* 2 1/4-20 5/8" screws<br />
* 4 1/4-20 1/4" screws<br />
<br />
The thermal subsystem includes all components to control heat flow into and out of the sample and monitor its temperature. Heating is accomplished using thermal electric coolers (TECs) in reverse. The hot side is placed in direct contact with the heating block, which is in direct contact with the sample vial. The TECs will be powered by manually controlling the Diablotek power supply. To avoid contamination of the sample or perturbation of its temperature, the temperature of a resistive temperature detector (RTD) embedded in the heating block will be monitored as a proxy for the sample temperature. In this part of the lab, air convection will cool down the sample when the Diablotek is switched off. The heat sink will help and the fan may be connected to a triple-output power supply to help, but these are included primarily for use in Part 2.<br />
<br />
===Parts===<br />
Gather the following components from the bins on the Center Cabinets, the DNA Melting drawer at the wet bench or on the East Cabinets:<br />
<br />
[[Image:CenterCabinets.jpg|right|250px|thumb|'''Center Cabinets''' The bins for most parts that you need will be set out on top of these cabinets, roughly in the center of the lab.]]<br />
[[Image:WetBench.jpg|right|250px|thumb|'''Wet Bench''' Supplies for our wet bench can be found in the three drawers at the left of the bench, the drawer left of the sink and on the wall behind and to the left as you face the wet bench. The drawer at the right of the wet bench contains various supplies for the DNA Melting instrument.]]<br />
[[Image:EastCabinets.jpg|right|250px|thumb|'''East Cabinets''' The center shelves of the East Cabinets contain the electronic breadboards, breadboard jumper wire, TEC connectors and TEC power supplies. Often these supplies are laid out on top of these cabinets.]]<br />
<br />
* These components are already assembled for you: A metal support bracket, having a fan, heatsink, 2 TEC heaters/coolers, and a heating block attached to it<br />
* 3 wire nuts<br />
* 1 Diablotek 250 W power supply (at your station or on a shelf above your station)<br />
* 1 red/black TEC extension wire assembly (in a box on the ''''Center Cabinets'''')<br />
<br />
===Assembly instructions===<br />
<br />
[[Image:TEC_picture.jpg|150 px|thumb|right|TEC showing hot side up when current flows into the red lead; i.e., when a positive voltage is applied between the red and black leads.]]<br />
<br />
# The bracket, TECs, fan and heatsink should already be assembled for you.<br />
#* The TECS are sandwiched between the heating block (on top) and the heat sink (underneath).<br />
# Verify that the colors of the leads match. If not, flip one TEC with reference to the picture at right.<br />
# Connect the black lead of the top TEC to the red lead of the bottom TEC using a wire nut.<br />
# Connect the TEC to the black/white (and black/yellow) extension wire assembly using wire nuts to join the white wire to the red lead of the top TEC and the black wire to the black lead on the bottom TEC, respectively.<br />
# Join the white connector on the assembly to the '''black''' 2x2 connector of the Diabloteck.<br />
#* Use only the '''black''' 2x2 connector, not the white one.<br />
# Test the TECs by touching the base of your heating block and turning the Diablotek on.<br />
#* The base should start to feel warm within 10-15 seconds.<br />
#* If the Diablotek immediately shuts off (the fan will stop spinning) then there is probably a short. Check all connections.<br />
# Once the TEC assembly wiring is checked, try the heating test again.<br />
# If it fails again, find an Instructor.<br />
<br />
==Optical subsystem==<br />
<br />
A good optical subsystem design will be compact, stable and simple. It includes the optical components of the excitation and fluorescence detection functional groups from the block diagram. A good design will efficiently illuminate the sample and collect the maximum possible amount of emitted fluorescent light. There are a variety of ways to construct the apparatus. Use the Instructor instrument(s), and the instructions below, as a guide. The set of instructions can help if you're not clear on how to replicate the example system in the lab. In addition to the following parts, you might want to add a few lenses to collimate your LED onto your sample, and to focus the light coming from your sample onto your photodiode. <br />
<br />
* 2 PH2 post holders<br />
* 2 BA1 post holder bases<br />
* 2 1/4-20 1/4" screws<br />
* 2 1/4-20 5/8" screws<br />
* 2 1/4" washers<br />
* 4 CP02 cage plates<br />
* 2 8-32 1/2" setscrews<br />
* 2 TR2 posts<br />
* 6 ER3 cage rails<br />
* 12 4-40 1/4" setscrews (or similar length)<br />
* 4 SML05 lens tubes<br />
* 4 SM1LTRR retaining rings<br />
* 1 blue LED PCB<br />
* 1 D470/40 excitation filter<br />
* 1 E515LPv2 emission filter<br />
* 1 SM1A6 adapter between SM1 and SM05 threads<br />
* 1 SM05PD1A mounted photodiode<br />
<br />
===Assembly instructions===<br />
Use an Instructor instrument or the Solid Works model above as guide to assemble your optical subassembly. Note also that these instructions are also a guid. If you wish to build your own personal design, please do.<br />
<br />
# Attach each PH2 to a BA1 using 1/4" screws.<br />
# Attach two of the CP02 cage plates to two TR2 posts using an 8-32 setscrew.<br />
# Insert the TR2/CP02 assembly into the PH2/BA1 assemblies and lock in place with the TS25H setscrews.<br />
# Insert three ER4 cage rails into each CP02 above and lock in place with SS4S025 set screws or similar.<br />
#* Leave the blank hole on the bottom of the assembly.<br />
# Attach one of the remaining CP02 cage plates on the free end of each assembly above and lock in place.<br />
# Attach each of the above assemblies to the MB1224 using a washer and 1/4-20 screw less than 1" in length.<br />
# Arrange on the MB1224 and adjust CP02 heights as desired.<br />
# Mount the LED PCB in an SML05 lens tube and fix with a retaining ring.<br />
# Mount the excitation and emission filters in each of two lens tubes and fix with retaining ring.<br />
#* The arrow label on the thickness of the filter points ''toward the light source''.<br />
# Thread the SM1A6 adapter into a lens tube and fix with a retaining ring.<br />
# Thread the photodiode into the SM1A6 adapter.<br />
#* Teflon pipe thread tape can be used to hold the photodiode in the desired position.<br />
# Mount each lens tube in the appropriate CP02 cage plates.<br />
# Arrange each the resultant assemblies on the breadboard to most fully illuminate the sample vial and to gather the most of the resultant light on the photodiode.<br />
<br />
==Electronics subsystem==<br />
<br />
The electrical subsystem includes the LED with a current-limiting resistor, the photodiode, the transimpedance amplifier and a temperature detection circuit. The remainder of this section provides an example of each of these systems and instructions to build and test each one. Nevertheless, alternate ideas are welcome. Feel free to discuss with the Instructors.<br />
<br />
===Parts===<br />
<br />
Gather the following components from the electronics drawer to the right of the optics drawers, from the counter top opposite the wet bench and from the cable organizer on the East wall to the right of the white board.<br />
* 1 electronic breadboard<br />
* 1 jump wire kit<br />
* 2 red 3' banana cable or similar<br />
* 1 black 3' banana cable or similar<br />
* individual wire, as-needed from the scrap wire box or the wire spools opposite the wet bench<br />
* 1 62 &Omega; resistor from the center counter top on the South wall<br />
* 1 15 k&Omega; resistor<br />
* other resistors, as-needed for the amplifier<br />
* 1 blue LED from the DNA Melting drawer at the far right of the wet bench<br />
* 2 LF411 op-amps from the electronics drawer<br />
* 4 capacitors between 0.1 and 22 uF from either the electronics drawer or the counter top where the screws are stored.<br />
<br />
===Assembly instructions===<br />
<br />
====LED circuit====<br />
The led circuit consists of a 62 &Omega; resistor in series with a blue, jumbo LED. The circuit will be powered by the 5 volt fixed output of the lab power supply. The 62 &Omega; resistor prevents excessive current from flowing through the LED. Excessive current will let the smoke out of the LED.<br />
<br />
# Build the LED circuit on your electronic breadboard.<br />
#* Don't forget to connect the resistor. Directly connecting the LED to 5 V will blow out the LED.<br />
# Disable the output of the lab power supply.<br />
# Connect the LED circuit to the fixed 5 V output.<br />
<br />
====Temperature detection circuit====<br />
A platinum RTD is embedded inside the heating block. The resistance of the RTD varies with temperature according to the equation ''R<sub>RTD</sub>''=1000 &Omega; + 3.85 ''&theta;'', where ''&theta;'' is the temperature of the RTD in degrees Celsius. The easiest way to make a temperature measurement is to connect the RTD in series with a fixed resistor and a power supply. The output voltage of the resulting divider circuit varies as a function of temperature. <br />
<br />
The maximum current of the RTD is 1 mA. You will use a 15 V supply to the circuit, so the resistor must be at least 14 k&Omega;. Use a 15 k&Omega; resistor.<br />
<br />
# '''Find an equation for ''V<sub>out</sub>'' as a function of temperature. What voltage do you expect given an input voltage of 15 V with the RTD at room temperature.'''<br />
# Turn off the output of the triple-output power supply.<br />
# Connect the RTD to the electronic breadboard.<br />
# '''Measure the resistance of the 15 k&Omega; resistor with a DMM and record the value in your lab report.'''<br />
# Connect the 15k&Omega; resistor in series with the RTD.<br />
# Set the power supply for '''SERIES''' operation.<br />
# Set the output voltage to 15 V on the supply and make note of its exact value using the DMM.<br />
# Set the current limit to 0.1 A.<br />
# Connect the positive terminal of '''CH1''' to a banana terminal on the breadboard for +15 V and wire it to the top red bus strip for +15 V supply.<br />
# Connect the negative terminal of '''CH2''' to a banana terminal on the breadboard for -15 V.<br />
# Connect the positive terminal of '''CH2''' to the black terminal on the breadboard for a ground reference and wire it to the top blue bus strip.<br />
#* Use three jump wires to minimize the parasitic voltage drop due to the breadboard tie points.<br />
# Connect the +15 V bus strip to the second vertical, red bus strip and wire this bus strip to the top of your 15 k&Omega; resistor.<br />
# Turn on the output of the power supply and measure the voltage across the RTD using the DMM.<br />
# '''What is the measured voltage of the RTD at room temperature and how does it compare to your predictions above?'''<br />
# '''What is the temperature, based on your measured voltage and how does it compare with the clock on the wall (or some better measure)?'''<br />
# '''How is the temperature calculation affected by a 1% change in the 15 V input voltage?'''<br />
<br />
====Photodiode amplifier circuit====<br />
<br />
The current produced by the photodiode detector is a few tens of nanoamperes. This very small current must be amplified to a level where it can be easily measured. The data acquisition system you will use can measure voltages between + and -10 V. Use the two-stage op amp circuit shown below to convert the photodiode current to a voltage.<br />
<br />
<center>[[Image:Part1_TransimpedanceAmplifier.jpg|700 px|thumb|center|Multi-stage photodiode amplification circuit]]</center><br />
<br />
You will have to decide how to distribute the gain between the two op amp stages in the circuit. It is best to put as much gain in early stages of a multi-stage amplifier as possible. However, when the gain of a single stage becomes very high, nonidealities of the op amp become bothersome. The first stage of the amplifier will behave reasonably well as long as the gain is less than about 10<sup>6</sup> &Omega;.<br />
<br />
# '''Analyze the photodiode amplifier circuit. Choose resistor values to achieve an overall gain that will yield an output of about 5 V when the input is 500 nA.'''<br />
# Construct the circuit on your breadboard.<br />
#* Disable the power supply before you build.<br />
#* Do not yet connect the photodiode.<br />
#* Orient the LF411 op-amps so that pin 1, which is marked with a dot on the package, is at the upper left.<br />
# Connect the +/- 15 V bus strips to power the op-amps power pins.<br />
# Connect the vertical blue bus strips to the horizontal blue bus strip.<br />
<br />
=====Test connectivity and basic operation=====<br />
<br />
# Verify that all your component connections are correct and match the design.<br />
# Check that wires and components are properly seated in the breadboard and not suspiciously loose.<br />
#* You can use the ohm-meter function of the digital multi-meter to verify continuity.<br />
# Turn on the power supply and verify that +15V appears on pin 7 and -15V on pin 4.<br />
# '''Disconnect the photodiode connection to pin 2 (the <math>v^-</math> input to the op-amp) and measure the output voltages of IC1 and IC2.'''<br />
#* With the photodiode disconnected, the current signal is zero. If you see a significant offset voltage (from 50 to 100 mV) at IC1 pin 6, then you may wish to add (or adjust the value of) <math>R_6</math>.<br />
#* Refer to [http://measure.mit.edu/~20.309/wiki/index.php?title=Real_electronics#Real_op-amps Real Electronics, real op-amps] or ask an instructor to understand and address this non-ideal behavior.<br />
<br />
==Data acquisition hardware subsystem==<br />
<br />
{{:20.309:DAQ System}}<br />
<br />
====Summary of DAQ inputs/outputs====<br />
<br />
A special connector has been prepared for you, connected as shown below. The connections and cable wire colors are also summarized [http://dl.dropbox.com/u/12957607/DAQ_Cable_Assembly_for_DNA_melting_lab.pdf in a memo].<br />
<br />
{| class="wikitable"<br />
|-<br />
! Signal Name<br />
! Signal Location<br />
! Ground Location<br />
! Pin wire color<br />
|-<br />
! DAQ Inputs <br />
|-<br />
! RTD<br />
| AI0+<br />
| AI0-<br />
| +Orange / -Black [lone pair, not with third (red) wire]<br />
|-<br />
! Photodiode <br />
| AI1+ <br />
| AI1-<br />
| +Green / -White<br />
|}<br />
<br />
<center>[[Image:DAQ_Wire_Colors.png|450 px|thumb|center|DAQ Connection Cable]]</center><br />
<br />
==DNAMelter software==<br />
<br />
[[Image:BasicDNAMelterIcon.png|75 px|left]]<br />
Use the <code>Basic DNA Melter GUI</code> program (located on the lab computer desktop) to collect data from the apparatus.<br />
<br />
At the end of an experimental run, use the "Save" button to write the date to a file. The data will be tab-delimited and can be read into Matlab with the <code>load</code> command.<br />
<br />
Documentation for the DNAMelter software is available here: [[DNA Melting: Using the Basic DNAMelter GUI]]<br />
<br />
====If you need to debug the DAQ (skip otherwise!)====<br />
* When debugging problems, it is frequently useful to ascertain whether the data acquisition system is working properly. National Instruments provides an application for this purpose called Measurement and Automation Explorer. Use Measurement and Automation Explorer to verify that the DAQ is properly connected to and communicating with the computer. The program also provides the ability to manually control all of the DAQ system's inputs and outputs.<br />
** Launch Measurement and Automation Explorer by selecting Start->All Applications->National Instruments->Measurement and Automation.<br />
** In the "Configuration" tab, select "Devices and Interfaces" and then "NI-DAQMx Devices." Select "Dev1."<br />
** If Dev 1 does not show up, the problem is that computer cannot communicate with the DAQ system. Ensure that the USB cable is connected to the DAQ system. If the cable is connected and the device still does not show up, unplug the USB cable, count to five, and plug it back in. <br />
** If the DAQ shows up under a different name, disconnect the USB cable. Delete all the "NI USB-6xxx" entries and then reconnect the USB cable. Verify that one entry reappears and that it is called "Dev1." <br />
** Select the "Test Panels" tab to manually control or read signals from the DAQ.<br />
* If you run DNAMelter and there is a data acquisition error, for example, "<code>Data acquisition cannot start!</code>", open Measurement and Automation Explorer and follow the steps above. Run DNAMelter again. If it still doesn't work, grab a beer or ask an instructor for help.<br />
* The DAQ has only one analog-to-digital converter (ADC). A multiplexer circuit sequentially samples voltages from the analog inputs configured in the control software. If any of the inputs is outside the allowable range of +/-10V, all of the inputs may behave erratically. The voltages reported may look reasonable so this can be very misleading. If none of the DAQ readings make sense, ensure that all of the analog voltages are in the allowable range and reset the DAQ by cycling the power of the DAQ (use the switch on the 6341 or disconnect the USB of the 6212).<br />
* If the DAQ gets short-circuited, it may crash. If this happens, Matlab will frequently give a deceptive error message, for example claiming that the limits on the band-pass filter do not satisfy some condition. In this case, correct the connection and cycle the DAQ power.<br />
<br />
==Part 1 measurements==<br />
<br />
Please read this entire section before beginning. Then start at Testing the instrument, with reference back to the sample sections as-needed.<br />
<br />
===Making samples===<br />
<br />
{{Template:Biohazard warning|message=LC Green in DMSO is readily absorbed through skin. Synthetic oligonucleotides may be harmful by inhalation, ingestion, or skin absorption. Wear gloves when handling samples. Wear safety goggles at all times when pipetting the LC Green/DNA samples. Do not create aerosols. The health effects of LC Green have not been thoroughly investigated. See the LC Green and synthetic oligonucleotide under <code>../EHS Guidelines/MSDS Repository</code> in the course locker for more information.}}<br />
<br />
====Sample prep steps====<br />
<br />
#Pipet 500 &mu;L of DNA plus dye solution into a glass vial. <br />
#Pipet up to 200 &mu;L of mineral oil on top of the sample to help prevent evaporation. It will help to pipette into the corners first. Be careful not to get oil on the cuvette sides far above the sample. It will run down during your experiment and cause shifts in the photodiode signal. Place the cap loosely on the vial. <br />
#*Keep the sample vertical to make sure the oil stays on top. <br />
<br />
* You can use the same sample for several heating/cooling cycles.<br />
* Only discard a sample if you lose significant volume due to evaporation or if your signal gets too low. <br />
<br />
To clean your glass vial between samples, flush with alcohol in the waste container and rinse with water at the drain. Suck out residual liquid with the vacuum and drawn Pasteur pipette to the left of the sink.<br />
<br />
====Sample disposal====<br />
<br />
{{Template:Environmental Warning|message=Discard pipette tips with DNA sample residue in the pipette tips or the ''Biohazard Sharps'' container. Do not pour synthetic oligonucleotides with LC Green down the drain. Pour your used samples into the waste container provided in the middle of the wet bench.}}<br />
<br />
===Testing the instrument===<br />
<br />
Once your apparatus is built, use samples of fluorescein (~ 30&mu;M concentration) as a stand-in for LC Green + DNA to test the instrument. Fluorescein is cheap, non-toxic and will not bleach as quickly as LC Green.<br />
<br />
====Making a simple measurement of your instrument's low frequency SNR====<br />
<br />
Measure the signal to noise ratio (SNR) of the instrument by comparing the difference between the average signal with a sample present and with water as a null reference. This process requires a vial containing 500 uL of 30 uM fluorescein and a vial containing the same amount of water. <br />
<br />
# Run the <code>Basic DNA Melter GUI</code>. <br />
# Place a vial of DI water in your instrument.<br />
# Clear the data and wait 10 seconds.<br />
# Replace the water vial with the DNA of fluorescein vial, record for another 10 seconds, then save the data. <br />
#* Be sure that all other conditions, such as temperature, are stable throughout the test. <br />
<br />
Compute the signal to noise ratio, <math>\text{SNR}=\frac{\langle V_{fluorescein} \rangle - \langle V_{water} \rangle}{\sigma_{fluorescein} }</math>, where <math>V_{fluorescein}</math> is the portion of the data recorded with a fluorescein sample, <math>V_{water}</math> is the portion of the signal corresponding to the water sample, and <math>\sigma_{fluorescein}</math> is the standard deviation of <math>V_{fluorescein}</math>.<br />
<br />
===Experiment steps===<br />
<br />
Once the instrument is confirmed to be functioning properly, follow these instructions to generate melting curve data for a 20 bp DNA and green dye solution.<br />
<br />
#Open and run the <code>Basic DNA Melter GUI</code> in Matlab and follow the instructions there.<br />
# Confirm that block temperature is near room temperature when the Diablotek supply is off.<br />
# Test a full heat/cool cycle with no DNA: Apply heat to the block by switching the Diablotek on to increase the block temperature to 95&deg;C, toggle the Diablotek power in order to hold the temperature long enough for the fluorescence curve to reach a minimum, then allow the block to cool down by switching off the Diablotek.<br />
#* Other approaches may be used. One goal would be to heat the block more slowly, possibly by toggling the current while heating.<br />
# Now prepare a sample using the stock of "beater" (possibly labeled as "junk") DNA. Don't worry, it melts just as well as your fresh samples will melt.<br />
# Place the vial in the instrument, but keep the LED off until ready to measure the melting curve.<br />
# Using the same temperature cycle protocol as in your test above, measure at least one melting curve of the beater DNA sample. Repeat as-desired to understand instrument operation.<br />
#* Be sure to save a copy in a local directory so you can plot it in your Part 1 report.<br />
<br />
{{:DNA Melting Report Requirements for Part 1}}<br />
<br />
==Lab manual sections==<br />
<br />
*[[Lab Manual:Measuring DNA Melting Curves]]<br />
*[[DNA Melting: Simulating DNA Melting - Basics]]<br />
*[[DNA Melting Part 1: Measuring Temperature and Fluorescence]]<br />
*[[DNA Melting Report Requirements for Part 1]]<br />
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]<br />
*[[DNA Melting Report Requirements for Part 2]]<br />
<br />
==References==<br />
<references/><br />
<br />
==Subset of datasheets==<br />
(Many more can be found online or on the course share)<br />
#[http://www.ni.com/pdf/manuals/371931f.pdf National Instruments USB-6212 user manual]<br />
#[http://www.ni.com/pdf/manuals/370784d.pdf National Instruments USB-6341 user manual]<br />
#[http://www.national.com/ds/LF/LF411.pdf Op-amp datasheet]<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/DNA_Melting_Part_1:_Measuring_Temperature_and_FluorescenceDNA Melting Part 1: Measuring Temperature and Fluorescence2015-11-10T19:44:37Z<p>Steven Nagle: /* Assembly instructions */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
<br />
[[Image:DNA Melting Block Diagram.jpg|frame|DNA melting apparatus block diagram showing the five functional groups of the instrument.]]<br />
<br />
<br />
''In theory, there is no difference between theory and practice. But, in practice, there is.'' <br />
<br />
''&mdash;[http://en.wikiquote.org/wiki/Jan_L._A._van_de_Snepscheut Jan L. A. van de Snepscheut]/[http://en.wikiquote.org/wiki/Yogi_Berra Yogi Berra]/and or [http://c2.com/cgi/wiki?DifferenceBetweenTheoryAndPractice Chuck Reid]'' <br />
<br />
You will construct a basic DNA melting instrument consisting of the five functional groups shown in the block diagram:<br />
* excitation<br />
* heating<br />
* fluorescent detection<br />
* temperature measurement<br />
* data acquisition and control<br />
<br />
A metal block will hold a sample vial in the excitation light path and transfer heat to the sample. The sample contains a combination of DNA, SYBR Green I and a salt. Recall that SYBR Green I fluoresces when it is bound to double stranded DNA (dsDNA) by a factor of up to 1000x compared to when it is bound to single stranded DNA (ssDNA). Detection of this fluorescence provides a relative measure of the fraction of dsDNA to total DNA, which can be compared to predictions from a thermodynamic model.<br />
<br />
The dye has a peak sensitivity to blue light at 497 nm so a blue LED will be used to illuminate the sample. The dye emits green light with an emission peak at 520 nm. A photodiode will be placed at 90&deg; to the LED source to detect the green light emitted by SYBR Green I. Because LEDs have a fairly broad spectrum, first a moderately narrow band-pass filter will be used to limit the excitation wavelength to between 450 and 490 nm. Then a complimentary long-pass emission filter will be placed between the sample and detector to pass only wavelengths longer than 515 nm. This filter combination, when used with this dye system, eliminates any background signal due to the excitation light.<br />
<br />
The sample block will be heated by a TEC (thermoelectric cooler) and its temperature measured by an embedded RTD (resistance temperature detector). Also, since photodiodes produce a very small amount of current, a high gain amplifier will convert the photodiode current into a measurable voltage. The DAQ (data acquisition card) and PC will digitize the amplified photodiode and RTD signals. A Matlab program is provided to record these fluorescence and temperature signals over time and save the data to a file. The file can then be loaded into Matlab for analysis, or if you prefer, Python or equivalent where the signals will be converted into dsDNA concentration and sample temperature. Finally, the dsDNA sample melting temperature will be estimated from a plot of df/dT vs T (''f'' represents the fraction of total DNA that is in double-stranded form) and from a comparison to the thermodynamic model.<br />
<br />
The functional groups in the block diagram are one of two fundamental frameworks used to describe an instrument. The other sub-divides the instrument into categories that often mirror historical areas of engineering. The former is focused on what will be done, including the overall scientific goals and the physical processes to be measured. The latter is focused on how it will be done, e.g. using optics, electronics or perhaps a practical knowledge of thermal management. A good engineer will constantly be thinking of the instrument in both frameworks. <br />
<br />
==Mechanical and thermal components==<br />
Mechanical support for the heating block, sample and optics will be built on a portable optical breadboard from [http://www.thorlabs.com ThorLabs]. Most of the components in the lab are from this supplier because the selection is large, the quality is quite good and the price reasonable. Although you are free to modify the design as the lab progresses, begin a build from the standard design shown above and demonstrated by the example in the lab. Gather the following Thorlabs components but do not start to assemble.<br />
<br />
* 1 MB1224 optical breadboard<br />
* 2 CL5 clamps (or similar)<br />
* 2 1/4-20 5/8" screws<br />
* 4 1/4-20 1/4" screws<br />
<br />
The thermal subsystem includes all components to control heat flow into and out of the sample and monitor its temperature. Heating is accomplished using thermal electric coolers (TECs) in reverse. The hot side is placed in direct contact with the heating block, which is in direct contact with the sample vial. The TECs will be powered by manually controlling the Diablotek power supply. To avoid contamination of the sample or perturbation of its temperature, the temperature of a resistive temperature detector (RTD) embedded in the heating block will be monitored as a proxy for the sample temperature. In this part of the lab, air convection will cool down the sample when the Diablotek is switched off. The heat sink will help and the fan may be connected to a triple-output power supply to help, but these are included primarily for use in Part 2.<br />
<br />
===Parts===<br />
Gather the following components from the bins on the Center Cabinets, the DNA Melting drawer at the wet bench or on the East Cabinets:<br />
<br />
[[Image:CenterCabinets.jpg|right|250px|thumb|'''Center Cabinets''' The bins for most parts that you need will be set out on top of these cabinets, roughly in the center of the lab.]]<br />
[[Image:WetBench.jpg|right|250px|thumb|'''Wet Bench''' Supplies for our wet bench can be found in the three drawers at the left of the bench, the drawer left of the sink and on the wall behind and to the left as you face the wet bench. The drawer at the right of the wet bench contains various supplies for the DNA Melting instrument.]]<br />
[[Image:EastCabinets.jpg|right|250px|thumb|'''East Cabinets''' The center shelves of the East Cabinets contain the electronic breadboards, breadboard jumper wire, TEC connectors and TEC power supplies. Often these supplies are laid out on top of these cabinets.]]<br />
<br />
* These components are already assembled for you: A metal support bracket, having a fan, heatsink, 2 TEC heaters/coolers, and a heating block attached to it<br />
* 3 wire nuts<br />
* 1 Diablotek 250 W power supply (at your station or on a shelf above your station)<br />
* 1 red/black TEC extension wire assembly (in a box on the ''''Center Cabinets'''')<br />
<br />
===Assembly instructions===<br />
<br />
[[Image:TEC_picture.jpg|150 px|thumb|right|TEC showing hot side up when current flows into the red lead; i.e., when a positive voltage is applied between the red and black leads.]]<br />
<br />
# The bracket, TECs, fan and heatsink should already be assembled for you.<br />
#* The TECS are sandwiched between the heating block (on top) and the heat sink (underneath).<br />
# Verify that the colors of the leads match. If not, flip one TEC with reference to the picture at right.<br />
# Connect the black lead of the top TEC to the red lead of the bottom TEC using a wire nut.<br />
# Connect the TEC to the black/white (and black/yellow) extension wire assembly using wire nuts to join the white wire to the red lead of the top TEC and the black wire to the black lead on the bottom TEC, respectively.<br />
# Join the white connector on the assembly to the '''black''' 2x2 connector of the Diabloteck.<br />
#* Use only the '''black''' 2x2 connector, not the white one.<br />
# Test the TECs by touching the base of your heating block and turning the Diablotek on.<br />
#* The base should start to feel warm within 10-15 seconds.<br />
#* If the Diablotek immediately shuts off (the fan will stop spinning) then there is probably a short. Check all connections.<br />
# Once the TEC assembly wiring is checked, try the heating test again.<br />
# If it fails again, find an Instructor.<br />
<br />
==Optical subsystem==<br />
<br />
A good optical subsystem design will be compact, stable and simple. It includes the optical components of the excitation and fluorescence detection functional groups from the block diagram. A good design will efficiently illuminate the sample and collect the maximum possible amount of emitted fluorescent light. There are a variety of ways to construct the apparatus. Use the Instructor instrument(s), and the instructions below, as a guide. The set of instructions can help if you're not clear on how to replicate the example system in the lab. In addition to the following parts, you might want to add a few lenses to collimate your LED onto your sample, and to focus the light coming from your sample onto your photodiode. <br />
<br />
* 2 PH2 post holders<br />
* 2 BA1 post holder bases<br />
* 2 1/4-20 1/4" screws<br />
* 2 1/4-20 5/8" screws<br />
* 2 1/4" washers<br />
* 4 CP02 cage plates<br />
* 2 8-32 1/2" setscrews<br />
* 2 TR2 posts<br />
* 6 ER3 cage rails<br />
* 12 4-40 1/4" setscrews (or similar length)<br />
* 4 SML05 lens tubes<br />
* 4 SM1LTRR retaining rings<br />
* 1 blue LED PCB<br />
* 1 D470/40 excitation filter<br />
* 1 E515LPv2 emission filter<br />
* 1 SM1A6 adapter between SM1 and SM05 threads<br />
* 1 SM05PD1A mounted photodiode<br />
<br />
===Assembly instructions===<br />
Use an Instructor instrument or the Solid Works model above as guide to assemble your optical subassembly. Note also that these instructions are also a guid. If you wish to build your own personal design, please do.<br />
<br />
# Attach each PH2 to a BA1 using 1/4" screws.<br />
# Attach two of the CP02 cage plates to two TR2 posts using an 8-32 setscrew.<br />
# Insert the TR2/CP02 assembly into the PH2/BA1 assemblies and lock in place with the TS25H setscrews.<br />
# Insert three ER4 cage rails into each CP02 above and lock in place with SS4S025 set screws or similar.<br />
#* Leave the blank hole on the bottom of the assembly.<br />
# Attach one of the remaining CP02 cage plates on the free end of each assembly above and lock in place.<br />
# Attach each of the above assemblies to the MB1224 using a washer and 1/4-20 screw less than 1" in length.<br />
# Arrange on the MB1224 and adjust CP02 heights as desired.<br />
# Mount the LED PCB in an SML05 lens tube and fix with a retaining ring.<br />
# Mount the excitation and emission filters in each of two lens tubes and fix with retaining ring.<br />
#* The arrow label on the thickness of the filter points ''toward the light source''.<br />
# Thread the SM1A6 adapter into a lens tube and fix with a retaining ring.<br />
# Thread the photodiode into the SM1A6 adapter.<br />
#* Teflon pipe thread tape can be used to hold the photodiode in the desired position.<br />
# Mount each lens tube in the appropriate CP02 cage plates.<br />
# Arrange each the resultant assemblies on the breadboard to most fully illuminate the sample vial and to gather the most of the resultant light on the photodiode.<br />
<br />
==Electronics subsystem==<br />
<br />
The electrical subsystem includes the LED with a current-limiting resistor, the photodiode, the transimpedance amplifier and a temperature detection circuit. The remainder of this section provides an example of each of these systems and instructions to build and test each one. Nevertheless, alternate ideas are welcome. Feel free to discuss with the Instructors.<br />
<br />
===Parts===<br />
<br />
Gather the following components from the electronics drawer to the right of the optics drawers, from the counter top opposite the wet bench and from the cable organizer on the East wall to the right of the white board.<br />
* 1 electronic breadboard<br />
* 1 jump wire kit<br />
* 2 red 3' banana cable or similar<br />
* 1 black 3' banana cable or similar<br />
* individual wire, as-needed from the scrap wire box or the wire spools opposite the wet bench<br />
* 1 62 &Omega; resistor from the center counter top on the South wall<br />
* 1 15 k&Omega; resistor<br />
* other resistors, as-needed for the amplifier<br />
* 1 blue LED from the DNA Melting drawer at the far right of the wet bench<br />
* 2 LF411 op-amps from the electronics drawer<br />
* 4 capacitors between 0.1 and 22 uF from either the electronics drawer or the counter top where the screws are stored.<br />
<br />
===Assembly instructions===<br />
<br />
====LED circuit====<br />
The led circuit consists of a 62 &Omega; resistor in series with a blue, jumbo LED. The circuit will be powered by the 5 volt fixed output of the lab power supply. The 62 &Omega; resistor prevents excessive current from flowing through the LED. Excessive current will let the smoke out of the LED.<br />
<br />
# Build the LED circuit on your electronic breadboard.<br />
#* Don't forget to connect the resistor. Directly connecting the LED to 5 V will blow out the LED.<br />
# Disable the output of the lab power supply.<br />
# Connect the LED circuit to the fixed 5 V output.<br />
<br />
====Temperature detection circuit====<br />
A platinum RTD is embedded inside the heating block. The resistance of the RTD varies with temperature according to the equation ''R<sub>RTD</sub>''=1000 &Omega; + 3.85 ''&theta;'', where ''&theta;'' is the temperature of the RTD in degrees Celsius. The easiest way to make a temperature measurement is to connect the RTD in series with a fixed resistor and a power supply. The output voltage of the resulting divider circuit varies as a function of temperature. <br />
<br />
The maximum current of the RTD is 1 mA. You will use a 15 V supply to the circuit, so the resistor must be at least 14 k&Omega;. Use a 15 k&Omega; resistor.<br />
<br />
# '''Find an equation for ''V<sub>out</sub>'' as a function of temperature. What voltage do you expect given an input voltage of 15 V with the RTD at room temperature.'''<br />
# Turn off the output of the triple-output power supply.<br />
# Connect the RTD to the electronic breadboard.<br />
# '''Measure the resistance of the 15 k&Omega; resistor with a DMM and record the value in your lab report.'''<br />
# Connect the 15k&Omega; resistor in series with the RTD.<br />
# Set the power supply for '''SERIES''' operation.<br />
# Set the output voltage to 15 V on the supply and make note of its exact value using the DMM.<br />
# Set the current limit to 0.1 A.<br />
# Connect the positive terminal of '''CH1''' to a banana terminal on the breadboard for +15 V and wire it to the top red bus strip for +15 V supply.<br />
# Connect the negative terminal of '''CH2''' to a banana terminal on the breadboard for -15 V.<br />
# Connect the positive terminal of '''CH2''' to the black terminal on the breadboard for a ground reference and wire it to the top blue bus strip.<br />
#* Use three jump wires to minimize the parasitic voltage drop due to the breadboard tie points.<br />
# Connect the +15 V bus strip to the second vertical, red bus strip and wire this bus strip to the top of your 15 k&Omega; resistor.<br />
# Turn on the output of the power supply and measure the voltage across the RTD using the DMM.<br />
# '''What is the measured voltage of the RTD at room temperature and how does it compare to your predictions above?'''<br />
# '''What is the temperature, based on your measured voltage and how does it compare with the clock on the wall (or some better measure)?'''<br />
# '''How is the temperature calculation affected by a 1% change in the 15 V input voltage?'''<br />
<br />
====Photodiode amplifier circuit====<br />
<br />
The current produced by the photodiode detector is a few tens of nanoamperes. This very small current must be amplified to a level where it can be easily measured. The data acquisition system you will use can measure voltages between + and -10 V. Use the two-stage op amp circuit shown below to convert the photodiode current to a voltage.<br />
<br />
<center>[[Image:Part1_TransimpedanceAmplifier.jpg|700 px|thumb|center|Multi-stage photodiode amplification circuit]]</center><br />
<br />
You will have to decide how to distribute the gain between the two op amp stages in the circuit. It is best to put as much gain in early stages of a multi-stage amplifier as possible. However, when the gain of a single stage becomes very high, nonidealities of the op amp become bothersome. The first stage of the amplifier will behave reasonably well as long as the gain is less than about 10<sup>6</sup> &Omega;.<br />
<br />
# '''Analyze the photodiode amplifier circuit. Choose resistor values to achieve an overall gain that will yield an output of about 5 V when the input is 500 nA.'''<br />
# Construct the circuit on your breadboard.<br />
#* Disable the power supply before you build.<br />
#* Do not yet connect the photodiode.<br />
#* Orient the LF411 op-amps so that pin 1, which is marked with a dot on the package, is at the upper left.<br />
# Connect the +/- 15 V bus strips to power the op-amps power pins.<br />
# Connect the vertical blue bus strips to the horizontal blue bus strip.<br />
<br />
=====Test connectivity and basic operation=====<br />
<br />
# Verify that all your component connections are correct and match the design.<br />
# Check that wires and components are properly seated in the breadboard and not suspiciously loose.<br />
#* You can use the ohm-meter function of the digital multi-meter to verify continuity.<br />
# Turn on the power supply and verify that +15V appears on pin 7 and -15V on pin 4.<br />
# '''Disconnect the photodiode connection to pin 2 (the <math>v^-</math> input to the op-amp) and measure the output voltages of IC1 and IC2.'''<br />
#* With the photodiode disconnected, the current signal is zero. If you see a significant offset voltage (from 50 to 100 mV) at IC1 pin 6, then you may wish to add (or adjust the value of) <math>R_6</math>.<br />
#* Refer to [http://measure.mit.edu/~20.309/wiki/index.php?title=Real_electronics#Real_op-amps Real Electronics, real op-amps] or ask an instructor to understand and address this non-ideal behavior.<br />
<br />
==Data acquisition hardware subsystem==<br />
<br />
{{:20.309:DAQ System}}<br />
<br />
====Summary of DAQ inputs/outputs====<br />
<br />
A special connector has been prepared for you, connected as shown below. The connections and cable wire colors are also summarized [http://dl.dropbox.com/u/12957607/DAQ_Cable_Assembly_for_DNA_melting_lab.pdf in a memo].<br />
<br />
{| class="wikitable"<br />
|-<br />
! Signal Name<br />
! Signal Location<br />
! Ground Location<br />
! Pin wire color<br />
|-<br />
! DAQ Inputs <br />
|-<br />
! RTD<br />
| AI0+<br />
| AI0-<br />
| +Orange / -Black [lone pair, not with third (red) wire]<br />
|-<br />
! Photodiode <br />
| AI1+ <br />
| AI1-<br />
| +Green / -White<br />
|}<br />
<br />
<center>[[Image:DAQ_Wire_Colors.png|450 px|thumb|center|DAQ Connection Cable]]</center><br />
<br />
==DNAMelter software==<br />
<br />
[[Image:BasicDNAMelterIcon.png|75 px|left]]<br />
Use the <code>Basic DNA Melter GUI</code> program (located on the lab computer desktop) to collect data from the apparatus.<br />
<br />
At the end of an experimental run, use the "Save" button to write the date to a file. The data will be tab-delimited and can be read into Matlab with the <code>load</code> command.<br />
<br />
Documentation for the DNAMelter software is available here: [[DNA Melting: Using the Basic DNAMelter GUI]]<br />
<br />
====If you need to debug the DAQ (skip otherwise!)====<br />
* When debugging problems, it is frequently useful to ascertain whether the data acquisition system is working properly. National Instruments provides an application for this purpose called Measurement and Automation Explorer. Use Measurement and Automation Explorer to verify that the DAQ is properly connected to and communicating with the computer. The program also provides the ability to manually control all of the DAQ system's inputs and outputs.<br />
** Launch Measurement and Automation Explorer by selecting Start->All Applications->National Instruments->Measurement and Automation.<br />
** In the "Configuration" tab, select "Devices and Interfaces" and then "NI-DAQMx Devices." Select "Dev1."<br />
** If Dev 1 does not show up, the problem is that computer cannot communicate with the DAQ system. Ensure that the USB cable is connected to the DAQ system. If the cable is connected and the device still does not show up, unplug the USB cable, count to five, and plug it back in. <br />
** If the DAQ shows up under a different name, disconnect the USB cable. Delete all the "NI USB-6xxx" entries and then reconnect the USB cable. Verify that one entry reappears and that it is called "Dev1." <br />
** Select the "Test Panels" tab to manually control or read signals from the DAQ.<br />
* If you run DNAMelter and there is a data acquisition error, for example, "<code>Data acquisition cannot start!</code>", open Measurement and Automation Explorer and follow the steps above. Run DNAMelter again. If it still doesn't work, grab a beer or ask an instructor for help.<br />
* The DAQ has only one analog-to-digital converter (ADC). A multiplexer circuit sequentially samples voltages from the analog inputs configured in the control software. If any of the inputs is outside the allowable range of +/-10V, all of the inputs may behave erratically. The voltages reported may look reasonable so this can be very misleading. If none of the DAQ readings make sense, ensure that all of the analog voltages are in the allowable range and reset the DAQ by cycling the power of the DAQ (use the switch on the 6341 or disconnect the USB of the 6212).<br />
* If the DAQ gets short-circuited, it may crash. If this happens, Matlab will frequently give a deceptive error message, for example claiming that the limits on the band-pass filter do not satisfy some condition. In this case, correct the connection and cycle the DAQ power.<br />
<br />
==Part 1 measurements==<br />
<br />
Please read this entire section before beginning. Then start at Testing the instrument, with reference back to the sample sections as-needed.<br />
<br />
===Making samples===<br />
<br />
{{Template:Biohazard warning|message=LC Green in DMSO is readily absorbed through skin. Synthetic oligonucleotides may be harmful by inhalation, ingestion, or skin absorption. Wear gloves when handling samples. Wear safety goggles at all times when pipetting the LC Green/DNA samples. Do not create aerosols. The health effects of LC Green have not been thoroughly investigated. See the LC Green and synthetic oligonucleotide under <code>../EHS Guidelines/MSDS Repository</code> in the course locker for more information.}}<br />
<br />
====Sample prep steps====<br />
<br />
#Pipet 500 &mu;L of DNA plus dye solution into a glass vial. <br />
#Pipet up to 200 &mu;L of mineral oil on top of the sample to help prevent evaporation. It will help to pipette into the corners first. Be careful not to get oil on the cuvette sides far above the sample. It will run down during your experiment and cause shifts in the photodiode signal. Place the cap loosely on the vial. <br />
#*Keep the sample vertical to make sure the oil stays on top. <br />
<br />
* You can use the same sample for several heating/cooling cycles.<br />
* Only discard a sample if you lose significant volume due to evaporation or if your signal gets too low. <br />
<br />
To clean your glass vial between samples, flush with alcohol in the waste container and rinse with water at the drain. Suck out residual liquid with the vacuum and drawn Pasteur pipette to the left of the sink.<br />
<br />
====Sample disposal====<br />
<br />
{{Template:Environmental Warning|message=Discard pipette tips with DNA sample residue in the pipette tips or the ''Biohazard Sharps'' container. Do not pour synthetic oligonucleotides with LC Green down the drain. Pour your used samples into the waste container provided in the middle of the wet bench.}}<br />
<br />
===Testing the instrument===<br />
<br />
Once your apparatus is built, use samples of fluorescein (~ 30&mu;M concentration) as a stand-in for LC Green + DNA to test the instrument. Fluorescein is cheap, non-toxic and will not bleach as quickly as LC Green.<br />
<br />
====Making a simple measurement of your instrument's low frequency SNR====<br />
<br />
Measure the signal to noise ratio (SNR) of the instrument by comparing the difference between the average signal with a sample present and with water as a null reference. This process requires a vial containing 500 uL of 30 uM fluorescein and a vial containing the same amount of water. <br />
<br />
# Run the <code>Basic DNA Melter GUI</code>. <br />
# Place a vial of DI water in your instrument.<br />
# Clear the data and wait 10 seconds.<br />
# Replace the water vial with the DNA of fluorescein vial, record for another 10 seconds, then save the data. <br />
#* Be sure that all other conditions, such as temperature, are stable throughout the test. <br />
<br />
Compute the signal to noise ratio, <math>\text{SNR}=\frac{\langle V_{fluorescein} \rangle - \langle V_{water} \rangle}{\sigma_{fluorescein} }</math>, where <math>V_{fluorescein}</math> is the portion of the data recorded with a fluorescein sample, <math>V_{water}</math> is the portion of the signal corresponding to the water sample, and <math>\sigma_{fluorescein}</math> is the standard deviation of <math>V_{fluorescein}</math>.<br />
<br />
===Experiment steps===<br />
<br />
Once the instrument is confirmed to be functioning properly, follow these instructions to generate melting curve data for the 20 bp DNA-LC Green solution.<br />
<br />
#Open and run the <code>Basic DNA Melter GUI</code> in Matlab and follow the instructions there.<br />
# Confirm that block temperature is near room temperature when the Diablotek supply is off.<br />
# Apply heat to the block by switching the Diablotek on to increase the block temperature to 95&deg;C, toggle the Diablotek power in order to hold the temperature long enough for the fluorescence curve to reach a minimum, then allow the block to cool down by switching off the Diablotek.<br />
#* Other approaches may be used. One goal would be to heat the block more slowly, possibly by toggling the current while heating.<br />
# Now prepare a sample using the stock of "beater" DNA.<br />
# Place the vial in the instrument, but keep the LED off until ready to measure the melting curve.<br />
# Measure melting curves of the beater DNA sample as-desired to understand instrument operation.<br />
# Prepare a sample using fresh 20 bp 100 mM KCl DNA.<br />
# Measure a melting curve of the fresh sample.<br />
#* Be sure to save a copy in a local directory.<br />
<br />
{{:DNA Melting Report Requirements for Part 1}}<br />
<br />
==Lab manual sections==<br />
<br />
*[[Lab Manual:Measuring DNA Melting Curves]]<br />
*[[DNA Melting: Simulating DNA Melting - Basics]]<br />
*[[DNA Melting Part 1: Measuring Temperature and Fluorescence]]<br />
*[[DNA Melting Report Requirements for Part 1]]<br />
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]<br />
*[[DNA Melting Report Requirements for Part 2]]<br />
<br />
==References==<br />
<references/><br />
<br />
==Subset of datasheets==<br />
(Many more can be found online or on the course share)<br />
#[http://www.ni.com/pdf/manuals/371931f.pdf National Instruments USB-6212 user manual]<br />
#[http://www.ni.com/pdf/manuals/370784d.pdf National Instruments USB-6341 user manual]<br />
#[http://www.national.com/ds/LF/LF411.pdf Op-amp datasheet]<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/DNA_Melting_Part_1:_Measuring_Temperature_and_FluorescenceDNA Melting Part 1: Measuring Temperature and Fluorescence2015-11-10T19:30:46Z<p>Steven Nagle: /* Assembly instructions */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
<br />
[[Image:DNA Melting Block Diagram.jpg|frame|DNA melting apparatus block diagram showing the five functional groups of the instrument.]]<br />
<br />
<br />
''In theory, there is no difference between theory and practice. But, in practice, there is.'' <br />
<br />
''&mdash;[http://en.wikiquote.org/wiki/Jan_L._A._van_de_Snepscheut Jan L. A. van de Snepscheut]/[http://en.wikiquote.org/wiki/Yogi_Berra Yogi Berra]/and or [http://c2.com/cgi/wiki?DifferenceBetweenTheoryAndPractice Chuck Reid]'' <br />
<br />
You will construct a basic DNA melting instrument consisting of the five functional groups shown in the block diagram:<br />
* excitation<br />
* heating<br />
* fluorescent detection<br />
* temperature measurement<br />
* data acquisition and control<br />
<br />
A metal block will hold a sample vial in the excitation light path and transfer heat to the sample. The sample contains a combination of DNA, SYBR Green I and a salt. Recall that SYBR Green I fluoresces when it is bound to double stranded DNA (dsDNA) by a factor of up to 1000x compared to when it is bound to single stranded DNA (ssDNA). Detection of this fluorescence provides a relative measure of the fraction of dsDNA to total DNA, which can be compared to predictions from a thermodynamic model.<br />
<br />
The dye has a peak sensitivity to blue light at 497 nm so a blue LED will be used to illuminate the sample. The dye emits green light with an emission peak at 520 nm. A photodiode will be placed at 90&deg; to the LED source to detect the green light emitted by SYBR Green I. Because LEDs have a fairly broad spectrum, first a moderately narrow band-pass filter will be used to limit the excitation wavelength to between 450 and 490 nm. Then a complimentary long-pass emission filter will be placed between the sample and detector to pass only wavelengths longer than 515 nm. This filter combination, when used with this dye system, eliminates any background signal due to the excitation light.<br />
<br />
The sample block will be heated by a TEC (thermoelectric cooler) and its temperature measured by an embedded RTD (resistance temperature detector). Also, since photodiodes produce a very small amount of current, a high gain amplifier will convert the photodiode current into a measurable voltage. The DAQ (data acquisition card) and PC will digitize the amplified photodiode and RTD signals. A Matlab program is provided to record these fluorescence and temperature signals over time and save the data to a file. The file can then be loaded into Matlab for analysis, or if you prefer, Python or equivalent where the signals will be converted into dsDNA concentration and sample temperature. Finally, the dsDNA sample melting temperature will be estimated from a plot of df/dT vs T (''f'' represents the fraction of total DNA that is in double-stranded form) and from a comparison to the thermodynamic model.<br />
<br />
The functional groups in the block diagram are one of two fundamental frameworks used to describe an instrument. The other sub-divides the instrument into categories that often mirror historical areas of engineering. The former is focused on what will be done, including the overall scientific goals and the physical processes to be measured. The latter is focused on how it will be done, e.g. using optics, electronics or perhaps a practical knowledge of thermal management. A good engineer will constantly be thinking of the instrument in both frameworks. <br />
<br />
==Mechanical and thermal components==<br />
Mechanical support for the heating block, sample and optics will be built on a portable optical breadboard from [http://www.thorlabs.com ThorLabs]. Most of the components in the lab are from this supplier because the selection is large, the quality is quite good and the price reasonable. Although you are free to modify the design as the lab progresses, begin a build from the standard design shown above and demonstrated by the example in the lab. Gather the following Thorlabs components but do not start to assemble.<br />
<br />
* 1 MB1224 optical breadboard<br />
* 2 CL5 clamps (or similar)<br />
* 2 1/4-20 5/8" screws<br />
* 4 1/4-20 1/4" screws<br />
<br />
The thermal subsystem includes all components to control heat flow into and out of the sample and monitor its temperature. Heating is accomplished using thermal electric coolers (TECs) in reverse. The hot side is placed in direct contact with the heating block, which is in direct contact with the sample vial. The TECs will be powered by manually controlling the Diablotek power supply. To avoid contamination of the sample or perturbation of its temperature, the temperature of a resistive temperature detector (RTD) embedded in the heating block will be monitored as a proxy for the sample temperature. In this part of the lab, air convection will cool down the sample when the Diablotek is switched off. The heat sink will help and the fan may be connected to a triple-output power supply to help, but these are included primarily for use in Part 2.<br />
<br />
===Parts===<br />
Gather the following components from the bins on the Center Cabinets, the DNA Melting drawer at the wet bench or on the East Cabinets:<br />
<br />
[[Image:CenterCabinets.jpg|right|250px|thumb|'''Center Cabinets''' The bins for most parts that you need will be set out on top of these cabinets, roughly in the center of the lab.]]<br />
[[Image:WetBench.jpg|right|250px|thumb|'''Wet Bench''' Supplies for our wet bench can be found in the three drawers at the left of the bench, the drawer left of the sink and on the wall behind and to the left as you face the wet bench. The drawer at the right of the wet bench contains various supplies for the DNA Melting instrument.]]<br />
[[Image:EastCabinets.jpg|right|250px|thumb|'''East Cabinets''' The center shelves of the East Cabinets contain the electronic breadboards, breadboard jumper wire, TEC connectors and TEC power supplies. Often these supplies are laid out on top of these cabinets.]]<br />
<br />
* These components are already assembled for you: A metal support bracket, having a fan, heatsink, 2 TEC heaters/coolers, and a heating block attached to it<br />
* 3 wire nuts<br />
* 1 Diablotek 250 W power supply (at your station or on a shelf above your station)<br />
* 1 red/black TEC extension wire assembly (in a box on the ''''Center Cabinets'''')<br />
<br />
===Assembly instructions===<br />
<br />
[[Image:TEC_picture.jpg|150 px|thumb|right|TEC showing hot side up when current flows into the red lead]]<br />
<br />
Ensure that the following have been done/attached to your metal bracket:<br />
# The bracket, TECs, fan and heatsink should already be assembled for you.<br />
#* The TECS are sandwiched between the heating block (on top) and the heat sink (underneath).<br />
# Verify that the colors of the leads match. If not, flip one TEC.<br />
# Connect the black lead of the top TEC to the red lead of the bottom TEC using a wire nut.<br />
# Connect the TEC to the black/white (and black/yellow) extension wire assembly using wire nuts to join the white wire to the red lead of the top TEC and the black wire to the black lead on the bottom TEC, respectively.<br />
# Join the white connector on the assembly to the '''black''' 2x2 connector of the Diabloteck.<br />
# Test the TECs by holding the them between thumb and finger while briefly flipping the switch on the Diablotek.<br />
#* One side should immediately heat up while the other immediately cools.<br />
#* The top side should be the hot side when the red wires extend down and to the '''left'''.<br />
#* If the Diablotek immediately shuts off (the fan will stop spinning) then there is a short. Check all connections.<br />
# Once the TEC assembly checks out, butter the bottom side of the assembly and place that side on the heat sink with the leads facing to the right.<br />
# Finally, apply thermal grease to the top side of the TEC assembly, place the heating block on top and anchor to the heat sink using the 1/2" long 4-40 screws and plastic washers.<br />
<br />
==Optical subsystem==<br />
<br />
A good optical subsystem design will be compact, stable and simple. It includes the optical components of the excitation and fluorescence detection functional groups from the block diagram. A good design will efficiently illuminate the sample and collect the maximum possible amount of emitted fluorescent light. There are a variety of ways to construct the apparatus. Use the Instructor instrument(s), and the instructions below, as a guide. The set of instructions can help if you're not clear on how to replicate the example system in the lab. In addition to the following parts, you might want to add a few lenses to collimate your LED onto your sample, and to focus the light coming from your sample onto your photodiode. <br />
<br />
* 2 PH2 post holders<br />
* 2 BA1 post holder bases<br />
* 2 1/4-20 1/4" screws<br />
* 2 1/4-20 5/8" screws<br />
* 2 1/4" washers<br />
* 4 CP02 cage plates<br />
* 2 8-32 1/2" setscrews<br />
* 2 TR2 posts<br />
* 6 ER3 cage rails<br />
* 12 4-40 1/4" setscrews (or similar length)<br />
* 4 SML05 lens tubes<br />
* 4 SM1LTRR retaining rings<br />
* 1 blue LED PCB<br />
* 1 D470/40 excitation filter<br />
* 1 E515LPv2 emission filter<br />
* 1 SM1A6 adapter between SM1 and SM05 threads<br />
* 1 SM05PD1A mounted photodiode<br />
<br />
===Assembly instructions===<br />
Use an Instructor instrument or the Solid Works model above as guide to assemble your optical subassembly. Note also that these instructions are also a guid. If you wish to build your own personal design, please do.<br />
<br />
# Attach each PH2 to a BA1 using 1/4" screws.<br />
# Attach two of the CP02 cage plates to two TR2 posts using an 8-32 setscrew.<br />
# Insert the TR2/CP02 assembly into the PH2/BA1 assemblies and lock in place with the TS25H setscrews.<br />
# Insert three ER4 cage rails into each CP02 above and lock in place with SS4S025 set screws or similar.<br />
#* Leave the blank hole on the bottom of the assembly.<br />
# Attach one of the remaining CP02 cage plates on the free end of each assembly above and lock in place.<br />
# Attach each of the above assemblies to the MB1224 using a washer and 1/4-20 screw less than 1" in length.<br />
# Arrange on the MB1224 and adjust CP02 heights as desired.<br />
# Mount the LED PCB in an SML05 lens tube and fix with a retaining ring.<br />
# Mount the excitation and emission filters in each of two lens tubes and fix with retaining ring.<br />
#* The arrow label on the thickness of the filter points ''toward the light source''.<br />
# Thread the SM1A6 adapter into a lens tube and fix with a retaining ring.<br />
# Thread the photodiode into the SM1A6 adapter.<br />
#* Teflon pipe thread tape can be used to hold the photodiode in the desired position.<br />
# Mount each lens tube in the appropriate CP02 cage plates.<br />
# Arrange each the resultant assemblies on the breadboard to most fully illuminate the sample vial and to gather the most of the resultant light on the photodiode.<br />
<br />
==Electronics subsystem==<br />
<br />
The electrical subsystem includes the LED with a current-limiting resistor, the photodiode, the transimpedance amplifier and a temperature detection circuit. The remainder of this section provides an example of each of these systems and instructions to build and test each one. Nevertheless, alternate ideas are welcome. Feel free to discuss with the Instructors.<br />
<br />
===Parts===<br />
<br />
Gather the following components from the electronics drawer to the right of the optics drawers, from the counter top opposite the wet bench and from the cable organizer on the East wall to the right of the white board.<br />
* 1 electronic breadboard<br />
* 1 jump wire kit<br />
* 2 red 3' banana cable or similar<br />
* 1 black 3' banana cable or similar<br />
* individual wire, as-needed from the scrap wire box or the wire spools opposite the wet bench<br />
* 1 62 &Omega; resistor from the center counter top on the South wall<br />
* 1 15 k&Omega; resistor<br />
* other resistors, as-needed for the amplifier<br />
* 1 blue LED from the DNA Melting drawer at the far right of the wet bench<br />
* 2 LF411 op-amps from the electronics drawer<br />
* 4 capacitors between 0.1 and 22 uF from either the electronics drawer or the counter top where the screws are stored.<br />
<br />
===Assembly instructions===<br />
<br />
====LED circuit====<br />
The led circuit consists of a 62 &Omega; resistor in series with a blue, jumbo LED. The circuit will be powered by the 5 volt fixed output of the lab power supply. The 62 &Omega; resistor prevents excessive current from flowing through the LED. Excessive current will let the smoke out of the LED.<br />
<br />
# Build the LED circuit on your electronic breadboard.<br />
#* Don't forget to connect the resistor. Directly connecting the LED to 5 V will blow out the LED.<br />
# Disable the output of the lab power supply.<br />
# Connect the LED circuit to the fixed 5 V output.<br />
<br />
====Temperature detection circuit====<br />
A platinum RTD is embedded inside the heating block. The resistance of the RTD varies with temperature according to the equation ''R<sub>RTD</sub>''=1000 &Omega; + 3.85 ''&theta;'', where ''&theta;'' is the temperature of the RTD in degrees Celsius. The easiest way to make a temperature measurement is to connect the RTD in series with a fixed resistor and a power supply. The output voltage of the resulting divider circuit varies as a function of temperature. <br />
<br />
The maximum current of the RTD is 1 mA. You will use a 15 V supply to the circuit, so the resistor must be at least 14 k&Omega;. Use a 15 k&Omega; resistor.<br />
<br />
# '''Find an equation for ''V<sub>out</sub>'' as a function of temperature. What voltage do you expect given an input voltage of 15 V with the RTD at room temperature.'''<br />
# Turn off the output of the triple-output power supply.<br />
# Connect the RTD to the electronic breadboard.<br />
# '''Measure the resistance of the 15 k&Omega; resistor with a DMM and record the value in your lab report.'''<br />
# Connect the 15k&Omega; resistor in series with the RTD.<br />
# Set the power supply for '''SERIES''' operation.<br />
# Set the output voltage to 15 V on the supply and make note of its exact value using the DMM.<br />
# Set the current limit to 0.1 A.<br />
# Connect the positive terminal of '''CH1''' to a banana terminal on the breadboard for +15 V and wire it to the top red bus strip for +15 V supply.<br />
# Connect the negative terminal of '''CH2''' to a banana terminal on the breadboard for -15 V.<br />
# Connect the positive terminal of '''CH2''' to the black terminal on the breadboard for a ground reference and wire it to the top blue bus strip.<br />
#* Use three jump wires to minimize the parasitic voltage drop due to the breadboard tie points.<br />
# Connect the +15 V bus strip to the second vertical, red bus strip and wire this bus strip to the top of your 15 k&Omega; resistor.<br />
# Turn on the output of the power supply and measure the voltage across the RTD using the DMM.<br />
# '''What is the measured voltage of the RTD at room temperature and how does it compare to your predictions above?'''<br />
# '''What is the temperature, based on your measured voltage and how does it compare with the clock on the wall (or some better measure)?'''<br />
# '''How is the temperature calculation affected by a 1% change in the 15 V input voltage?'''<br />
<br />
====Photodiode amplifier circuit====<br />
<br />
The current produced by the photodiode detector is a few tens of nanoamperes. This very small current must be amplified to a level where it can be easily measured. The data acquisition system you will use can measure voltages between + and -10 V. Use the two-stage op amp circuit shown below to convert the photodiode current to a voltage.<br />
<br />
<center>[[Image:Part1_TransimpedanceAmplifier.jpg|700 px|thumb|center|Multi-stage photodiode amplification circuit]]</center><br />
<br />
You will have to decide how to distribute the gain between the two op amp stages in the circuit. It is best to put as much gain in early stages of a multi-stage amplifier as possible. However, when the gain of a single stage becomes very high, nonidealities of the op amp become bothersome. The first stage of the amplifier will behave reasonably well as long as the gain is less than about 10<sup>6</sup> &Omega;.<br />
<br />
# '''Analyze the photodiode amplifier circuit. Choose resistor values to achieve an overall gain that will yield an output of about 5 V when the input is 500 nA.'''<br />
# Construct the circuit on your breadboard.<br />
#* Disable the power supply before you build.<br />
#* Do not yet connect the photodiode.<br />
#* Orient the LF411 op-amps so that pin 1, which is marked with a dot on the package, is at the upper left.<br />
# Connect the +/- 15 V bus strips to power the op-amps power pins.<br />
# Connect the vertical blue bus strips to the horizontal blue bus strip.<br />
<br />
=====Test connectivity and basic operation=====<br />
<br />
# Verify that all your component connections are correct and match the design.<br />
# Check that wires and components are properly seated in the breadboard and not suspiciously loose.<br />
#* You can use the ohm-meter function of the digital multi-meter to verify continuity.<br />
# Turn on the power supply and verify that +15V appears on pin 7 and -15V on pin 4.<br />
# '''Disconnect the photodiode connection to pin 2 (the <math>v^-</math> input to the op-amp) and measure the output voltages of IC1 and IC2.'''<br />
#* With the photodiode disconnected, the current signal is zero. If you see a significant offset voltage (from 50 to 100 mV) at IC1 pin 6, then you may wish to add (or adjust the value of) <math>R_6</math>.<br />
#* Refer to [http://measure.mit.edu/~20.309/wiki/index.php?title=Real_electronics#Real_op-amps Real Electronics, real op-amps] or ask an instructor to understand and address this non-ideal behavior.<br />
<br />
==Data acquisition hardware subsystem==<br />
<br />
{{:20.309:DAQ System}}<br />
<br />
====Summary of DAQ inputs/outputs====<br />
<br />
A special connector has been prepared for you, connected as shown below. The connections and cable wire colors are also summarized [http://dl.dropbox.com/u/12957607/DAQ_Cable_Assembly_for_DNA_melting_lab.pdf in a memo].<br />
<br />
{| class="wikitable"<br />
|-<br />
! Signal Name<br />
! Signal Location<br />
! Ground Location<br />
! Pin wire color<br />
|-<br />
! DAQ Inputs <br />
|-<br />
! RTD<br />
| AI0+<br />
| AI0-<br />
| +Orange / -Black [lone pair, not with third (red) wire]<br />
|-<br />
! Photodiode <br />
| AI1+ <br />
| AI1-<br />
| +Green / -White<br />
|}<br />
<br />
<center>[[Image:DAQ_Wire_Colors.png|450 px|thumb|center|DAQ Connection Cable]]</center><br />
<br />
==DNAMelter software==<br />
<br />
[[Image:BasicDNAMelterIcon.png|75 px|left]]<br />
Use the <code>Basic DNA Melter GUI</code> program (located on the lab computer desktop) to collect data from the apparatus.<br />
<br />
At the end of an experimental run, use the "Save" button to write the date to a file. The data will be tab-delimited and can be read into Matlab with the <code>load</code> command.<br />
<br />
Documentation for the DNAMelter software is available here: [[DNA Melting: Using the Basic DNAMelter GUI]]<br />
<br />
====If you need to debug the DAQ (skip otherwise!)====<br />
* When debugging problems, it is frequently useful to ascertain whether the data acquisition system is working properly. National Instruments provides an application for this purpose called Measurement and Automation Explorer. Use Measurement and Automation Explorer to verify that the DAQ is properly connected to and communicating with the computer. The program also provides the ability to manually control all of the DAQ system's inputs and outputs.<br />
** Launch Measurement and Automation Explorer by selecting Start->All Applications->National Instruments->Measurement and Automation.<br />
** In the "Configuration" tab, select "Devices and Interfaces" and then "NI-DAQMx Devices." Select "Dev1."<br />
** If Dev 1 does not show up, the problem is that computer cannot communicate with the DAQ system. Ensure that the USB cable is connected to the DAQ system. If the cable is connected and the device still does not show up, unplug the USB cable, count to five, and plug it back in. <br />
** If the DAQ shows up under a different name, disconnect the USB cable. Delete all the "NI USB-6xxx" entries and then reconnect the USB cable. Verify that one entry reappears and that it is called "Dev1." <br />
** Select the "Test Panels" tab to manually control or read signals from the DAQ.<br />
* If you run DNAMelter and there is a data acquisition error, for example, "<code>Data acquisition cannot start!</code>", open Measurement and Automation Explorer and follow the steps above. Run DNAMelter again. If it still doesn't work, grab a beer or ask an instructor for help.<br />
* The DAQ has only one analog-to-digital converter (ADC). A multiplexer circuit sequentially samples voltages from the analog inputs configured in the control software. If any of the inputs is outside the allowable range of +/-10V, all of the inputs may behave erratically. The voltages reported may look reasonable so this can be very misleading. If none of the DAQ readings make sense, ensure that all of the analog voltages are in the allowable range and reset the DAQ by cycling the power of the DAQ (use the switch on the 6341 or disconnect the USB of the 6212).<br />
* If the DAQ gets short-circuited, it may crash. If this happens, Matlab will frequently give a deceptive error message, for example claiming that the limits on the band-pass filter do not satisfy some condition. In this case, correct the connection and cycle the DAQ power.<br />
<br />
==Part 1 measurements==<br />
<br />
Please read this entire section before beginning. Then start at Testing the instrument, with reference back to the sample sections as-needed.<br />
<br />
===Making samples===<br />
<br />
{{Template:Biohazard warning|message=LC Green in DMSO is readily absorbed through skin. Synthetic oligonucleotides may be harmful by inhalation, ingestion, or skin absorption. Wear gloves when handling samples. Wear safety goggles at all times when pipetting the LC Green/DNA samples. Do not create aerosols. The health effects of LC Green have not been thoroughly investigated. See the LC Green and synthetic oligonucleotide under <code>../EHS Guidelines/MSDS Repository</code> in the course locker for more information.}}<br />
<br />
====Sample prep steps====<br />
<br />
#Pipet 500 &mu;L of DNA plus dye solution into a glass vial. <br />
#Pipet up to 200 &mu;L of mineral oil on top of the sample to help prevent evaporation. It will help to pipette into the corners first. Be careful not to get oil on the cuvette sides far above the sample. It will run down during your experiment and cause shifts in the photodiode signal. Place the cap loosely on the vial. <br />
#*Keep the sample vertical to make sure the oil stays on top. <br />
<br />
* You can use the same sample for several heating/cooling cycles.<br />
* Only discard a sample if you lose significant volume due to evaporation or if your signal gets too low. <br />
<br />
To clean your glass vial between samples, flush with alcohol in the waste container and rinse with water at the drain. Suck out residual liquid with the vacuum and drawn Pasteur pipette to the left of the sink.<br />
<br />
====Sample disposal====<br />
<br />
{{Template:Environmental Warning|message=Discard pipette tips with DNA sample residue in the pipette tips or the ''Biohazard Sharps'' container. Do not pour synthetic oligonucleotides with LC Green down the drain. Pour your used samples into the waste container provided in the middle of the wet bench.}}<br />
<br />
===Testing the instrument===<br />
<br />
Once your apparatus is built, use samples of fluorescein (~ 30&mu;M concentration) as a stand-in for LC Green + DNA to test the instrument. Fluorescein is cheap, non-toxic and will not bleach as quickly as LC Green.<br />
<br />
====Making a simple measurement of your instrument's low frequency SNR====<br />
<br />
Measure the signal to noise ratio (SNR) of the instrument by comparing the difference between the average signal with a sample present and with water as a null reference. This process requires a vial containing 500 uL of 30 uM fluorescein and a vial containing the same amount of water. <br />
<br />
# Run the <code>Basic DNA Melter GUI</code>. <br />
# Place a vial of DI water in your instrument.<br />
# Clear the data and wait 10 seconds.<br />
# Replace the water vial with the DNA of fluorescein vial, record for another 10 seconds, then save the data. <br />
#* Be sure that all other conditions, such as temperature, are stable throughout the test. <br />
<br />
Compute the signal to noise ratio, <math>\text{SNR}=\frac{\langle V_{fluorescein} \rangle - \langle V_{water} \rangle}{\sigma_{fluorescein} }</math>, where <math>V_{fluorescein}</math> is the portion of the data recorded with a fluorescein sample, <math>V_{water}</math> is the portion of the signal corresponding to the water sample, and <math>\sigma_{fluorescein}</math> is the standard deviation of <math>V_{fluorescein}</math>.<br />
<br />
===Experiment steps===<br />
<br />
Once the instrument is confirmed to be functioning properly, follow these instructions to generate melting curve data for the 20 bp DNA-LC Green solution.<br />
<br />
#Open and run the <code>Basic DNA Melter GUI</code> in Matlab and follow the instructions there.<br />
# Confirm that block temperature is near room temperature when the Diablotek supply is off.<br />
# Apply heat to the block by switching the Diablotek on to increase the block temperature to 95&deg;C, toggle the Diablotek power in order to hold the temperature long enough for the fluorescence curve to reach a minimum, then allow the block to cool down by switching off the Diablotek.<br />
#* Other approaches may be used. One goal would be to heat the block more slowly, possibly by toggling the current while heating.<br />
# Now prepare a sample using the stock of "beater" DNA.<br />
# Place the vial in the instrument, but keep the LED off until ready to measure the melting curve.<br />
# Measure melting curves of the beater DNA sample as-desired to understand instrument operation.<br />
# Prepare a sample using fresh 20 bp 100 mM KCl DNA.<br />
# Measure a melting curve of the fresh sample.<br />
#* Be sure to save a copy in a local directory.<br />
<br />
{{:DNA Melting Report Requirements for Part 1}}<br />
<br />
==Lab manual sections==<br />
<br />
*[[Lab Manual:Measuring DNA Melting Curves]]<br />
*[[DNA Melting: Simulating DNA Melting - Basics]]<br />
*[[DNA Melting Part 1: Measuring Temperature and Fluorescence]]<br />
*[[DNA Melting Report Requirements for Part 1]]<br />
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]<br />
*[[DNA Melting Report Requirements for Part 2]]<br />
<br />
==References==<br />
<references/><br />
<br />
==Subset of datasheets==<br />
(Many more can be found online or on the course share)<br />
#[http://www.ni.com/pdf/manuals/371931f.pdf National Instruments USB-6212 user manual]<br />
#[http://www.ni.com/pdf/manuals/370784d.pdf National Instruments USB-6341 user manual]<br />
#[http://www.national.com/ds/LF/LF411.pdf Op-amp datasheet]<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/DNA_Melting_Part_1:_Measuring_Temperature_and_FluorescenceDNA Melting Part 1: Measuring Temperature and Fluorescence2015-11-08T21:41:06Z<p>Steven Nagle: /* Test connectivity and basic operation */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
<br />
[[Image:DNA Melting Block Diagram.jpg|frame|DNA melting apparatus block diagram showing the five functional groups of the instrument.]]<br />
<br />
<br />
''In theory, there is no difference between theory and practice. But, in practice, there is.'' <br />
<br />
''&mdash;[http://en.wikiquote.org/wiki/Jan_L._A._van_de_Snepscheut Jan L. A. van de Snepscheut]/[http://en.wikiquote.org/wiki/Yogi_Berra Yogi Berra]/and or [http://c2.com/cgi/wiki?DifferenceBetweenTheoryAndPractice Chuck Reid]'' <br />
<br />
You will construct a basic DNA melting instrument consisting of the five functional groups shown in the block diagram:<br />
* excitation<br />
* heating<br />
* fluorescent detection<br />
* temperature measurement<br />
* data acquisition and control<br />
<br />
A metal block will hold a sample vial in the excitation light path and transfer heat to the sample. The sample contains a combination of DNA, SYBR Green I and a salt. Recall that SYBR Green I fluoresces when it is bound to double stranded DNA (dsDNA) by a factor of up to 1000x compared to when it is bound to single stranded DNA (ssDNA). Detection of this fluorescence provides a relative measure of the fraction of dsDNA to total DNA, which can be compared to predictions from a thermodynamic model.<br />
<br />
The dye has a peak sensitivity to blue light at 497 nm so a blue LED will be used to illuminate the sample. The dye emits green light with an emission peak at 520 nm. A photodiode will be placed at 90&deg; to the LED source to detect the green light emitted by SYBR Green I. Because LEDs have a fairly broad spectrum, first a moderately narrow band-pass filter will be used to limit the excitation wavelength to between 450 and 490 nm. Then a complimentary long-pass emission filter will be placed between the sample and detector to pass only wavelengths longer than 515 nm. This filter combination, when used with this dye system, eliminates any background signal due to the excitation light.<br />
<br />
The sample block will be heated by a TEC (thermoelectric cooler) and its temperature measured by an embedded RTD (resistance temperature detector). Also, since photodiodes produce a very small amount of current, a high gain amplifier will convert the photodiode current into a measurable voltage. The DAQ (data acquisition card) and PC will digitize the amplified photodiode and RTD signals. A Matlab program is provided to record these fluorescence and temperature signals over time and save the data to a file. The file can then be loaded into Matlab for analysis, or if you prefer, Python or equivalent where the signals will be converted into dsDNA concentration and sample temperature. Finally, the dsDNA sample melting temperature will be estimated from a plot of df/dT vs T (''f'' represents the fraction of total DNA that is in double-stranded form) and from a comparison to the thermodynamic model.<br />
<br />
The functional groups in the block diagram are one of two fundamental frameworks used to describe an instrument. The other sub-divides the instrument into categories that often mirror historical areas of engineering. The former is focused on what will be done, including the overall scientific goals and the physical processes to be measured. The latter is focused on how it will be done, e.g. using optics, electronics or perhaps a practical knowledge of thermal management. A good engineer will constantly be thinking of the instrument in both frameworks. <br />
<br />
==Mechanical and thermal components==<br />
Mechanical support for the heating block, sample and optics will be built on a portable optical breadboard from [http://www.thorlabs.com ThorLabs]. Most of the components in the lab are from this supplier because the selection is large, the quality is quite good and the price reasonable. Although you are free to modify the design as the lab progresses, begin a build from the standard design shown above and demonstrated by the example in the lab. Gather the following Thorlabs components but do not start to assemble.<br />
<br />
* 1 MB1224 optical breadboard<br />
* 2 CL5 clamps (or similar)<br />
* 2 1/4-20 5/8" screws<br />
* 4 1/4-20 1/4" screws<br />
<br />
The thermal subsystem includes all components to control heat flow into and out of the sample and monitor its temperature. Heating is accomplished using thermal electric coolers (TECs) in reverse. The hot side is placed in direct contact with the heating block, which is in direct contact with the sample vial. The TECs will be powered by manually controlling the Diablotek power supply. To avoid contamination of the sample or perturbation of its temperature, the temperature of a resistive temperature detector (RTD) embedded in the heating block will be monitored as a proxy for the sample temperature. In this part of the lab, air convection will cool down the sample when the Diablotek is switched off. The heat sink will help and the fan may be connected to a triple-output power supply to help, but these are included primarily for use in Part 2.<br />
<br />
===Parts===<br />
Gather the following components from the bins on the Center Cabinets, the DNA Melting drawer at the wet bench or on the East Cabinets:<br />
<br />
[[Image:CenterCabinets.jpg|right|250px|thumb|'''Center Cabinets''' The bins for most parts that you need will be set out on top of these cabinets, roughly in the center of the lab.]]<br />
[[Image:WetBench.jpg|right|250px|thumb|'''Wet Bench''' Supplies for our wet bench can be found in the three drawers at the left of the bench, the drawer left of the sink and on the wall behind and to the left as you face the wet bench. The drawer at the right of the wet bench contains various supplies for the DNA Melting instrument.]]<br />
[[Image:EastCabinets.jpg|right|250px|thumb|'''East Cabinets''' The center shelves of the East Cabinets contain the electronic breadboards, breadboard jumper wire, TEC connectors and TEC power supplies. Often these supplies are laid out on top of these cabinets.]]<br />
<br />
* These components are already assembled for you: A metal support bracket, having a fan, heatsink, 2 TEC heaters/coolers, and a heating block attached to it<br />
* 3 wire nuts<br />
* 1 Diablotek 250 W power supply (at your station or on a shelf above your station)<br />
* 1 red/black TEC extension wire assembly (in a box on the ''''Center Cabinets'''')<br />
<br />
===Assembly instructions===<br />
<br />
[[Image:TEC_picture.jpg|150 px|thumb|right|TEC showing hot side up when current flows into the red lead]]<br />
<br />
Ensure that the following have been done/attached to your metal bracket:<br />
# The bracket, TECs, fan and heatsink should already be assembled for you.<br />
# Verify that the colors of the leads match. If not, flip one TEC.<br />
# Connect the black lead of the top TEC to the red lead of the bottom TEC using a wire nut.<br />
# Connect the TEC to the extension wire assembly using a wire nut to join the white wire to the red lead of the top TEC and the black wire to the black lead on the bottom TEC.<br />
# Join the white connector on the assembly to the black 2x2 connector of the Diabloteck.<br />
# Test the TECs by holding the them between thumb and finger while briefly flipping the switch on the Diablotek.<br />
#* One side should immediately heat up while the other immediately cools.<br />
#* The top side should be the hot side when the red wires extend down and to the '''left'''.<br />
#* If the Diablotek immediately shuts off (the fan will stop spinning) then there is a short. Check all connections.<br />
# Once the TEC assembly checks out, butter the bottom side of the assembly and place that side on the heat sink with the leads facing to the right.<br />
# Finally, apply thermal grease to the top side of the TEC assembly, place the heating block on top and anchor to the heat sink using the 1/2" long 4-40 screws and plastic washers.<br />
<br />
==Optical subsystem==<br />
<br />
A good optical subsystem design will be compact, stable and simple. It includes the optical components of the excitation and fluorescence detection functional groups from the block diagram. A good design will efficiently illuminate the sample and collect the maximum possible amount of emitted fluorescent light. There are a variety of ways to construct the apparatus. Use the Instructor instrument(s), and the instructions below, as a guide. The set of instructions can help if you're not clear on how to replicate the example system in the lab. In addition to the following parts, you might want to add a few lenses to collimate your LED onto your sample, and to focus the light coming from your sample onto your photodiode. <br />
<br />
* 2 PH2 post holders<br />
* 2 BA1 post holder bases<br />
* 2 1/4-20 1/4" screws<br />
* 2 1/4-20 5/8" screws<br />
* 2 1/4" washers<br />
* 4 CP02 cage plates<br />
* 2 8-32 1/2" setscrews<br />
* 2 TR2 posts<br />
* 6 ER3 cage rails<br />
* 12 4-40 1/4" setscrews (or similar length)<br />
* 4 SML05 lens tubes<br />
* 4 SM1LTRR retaining rings<br />
* 1 blue LED PCB<br />
* 1 D470/40 excitation filter<br />
* 1 E515LPv2 emission filter<br />
* 1 SM1A6 adapter between SM1 and SM05 threads<br />
* 1 SM05PD1A mounted photodiode<br />
<br />
===Assembly instructions===<br />
Use an Instructor instrument or the Solid Works model above as guide to assemble your optical subassembly. Note also that these instructions are also a guid. If you wish to build your own personal design, please do.<br />
<br />
# Attach each PH2 to a BA1 using 1/4" screws.<br />
# Attach two of the CP02 cage plates to two TR2 posts using an 8-32 setscrew.<br />
# Insert the TR2/CP02 assembly into the PH2/BA1 assemblies and lock in place with the TS25H setscrews.<br />
# Insert three ER4 cage rails into each CP02 above and lock in place with SS4S025 set screws or similar.<br />
#* Leave the blank hole on the bottom of the assembly.<br />
# Attach one of the remaining CP02 cage plates on the free end of each assembly above and lock in place.<br />
# Attach each of the above assemblies to the MB1224 using a washer and 1/4-20 screw less than 1" in length.<br />
# Arrange on the MB1224 and adjust CP02 heights as desired.<br />
# Mount the LED PCB in an SML05 lens tube and fix with a retaining ring.<br />
# Mount the excitation and emission filters in each of two lens tubes and fix with retaining ring.<br />
#* The arrow label on the thickness of the filter points ''toward the light source''.<br />
# Thread the SM1A6 adapter into a lens tube and fix with a retaining ring.<br />
# Thread the photodiode into the SM1A6 adapter.<br />
#* Teflon pipe thread tape can be used to hold the photodiode in the desired position.<br />
# Mount each lens tube in the appropriate CP02 cage plates.<br />
# Arrange each the resultant assemblies on the breadboard to most fully illuminate the sample vial and to gather the most of the resultant light on the photodiode.<br />
<br />
==Electronics subsystem==<br />
<br />
The electrical subsystem includes the LED with a current-limiting resistor, the photodiode, the transimpedance amplifier and a temperature detection circuit. The remainder of this section provides an example of each of these systems and instructions to build and test each one. Nevertheless, alternate ideas are welcome. Feel free to discuss with the Instructors.<br />
<br />
===Parts===<br />
<br />
Gather the following components from the electronics drawer to the right of the optics drawers, from the counter top opposite the wet bench and from the cable organizer on the East wall to the right of the white board.<br />
* 1 electronic breadboard<br />
* 1 jump wire kit<br />
* 2 red 3' banana cable or similar<br />
* 1 black 3' banana cable or similar<br />
* individual wire, as-needed from the scrap wire box or the wire spools opposite the wet bench<br />
* 1 62 &Omega; resistor from the center counter top on the South wall<br />
* 1 15 k&Omega; resistor<br />
* other resistors, as-needed for the amplifier<br />
* 1 blue LED from the DNA Melting drawer at the far right of the wet bench<br />
* 2 LF411 op-amps from the electronics drawer<br />
* 4 capacitors between 0.1 and 22 uF from either the electronics drawer or the counter top where the screws are stored.<br />
<br />
===Assembly instructions===<br />
<br />
====LED circuit====<br />
The led circuit consists of a 62 &Omega; resistor in series with a blue, jumbo LED. The circuit will be powered by the 5 volt fixed output of the lab power supply. The 62 &Omega; resistor prevents excessive current from flowing through the LED. Excessive current will let the smoke out of the LED.<br />
<br />
# Build the LED circuit on your electronic breadboard.<br />
#* Don't forget to connect the resistor. Directly connecting the LED to 5 V will blow out the LED.<br />
# Disable the output of the lab power supply.<br />
# Connect the LED circuit to the fixed 5 V output.<br />
<br />
====Temperature detection circuit====<br />
A platinum RTD is embedded inside the heating block. The resistance of the RTD varies with temperature according to the equation ''R<sub>RTD</sub>''=1000 &Omega; + 3.85 ''&theta;'', where ''&theta;'' is the temperature of the RTD in degrees Celsius. The easiest way to make a temperature measurement is to connect the RTD in series with a fixed resistor and a power supply. The output voltage of the resulting divider circuit varies as a function of temperature. <br />
<br />
The maximum current of the RTD is 1 mA. You will use a 15 V supply to the circuit, so the resistor must be at least 14 k&Omega;. Use a 15 k&Omega; resistor.<br />
<br />
# '''Find an equation for ''V<sub>out</sub>'' as a function of temperature. What voltage do you expect given an input voltage of 15 V with the RTD at room temperature.'''<br />
# Turn off the output of the triple-output power supply.<br />
# Connect the RTD to the electronic breadboard.<br />
# '''Measure the resistance of the 15 k&Omega; resistor with a DMM and record the value in your lab report.'''<br />
# Connect the 15k&Omega; resistor in series with the RTD.<br />
# Set the power supply for '''SERIES''' operation.<br />
# Set the output voltage to 15 V on the supply and make note of its exact value using the DMM.<br />
# Set the current limit to 0.1 A.<br />
# Connect the positive terminal of '''CH1''' to a banana terminal on the breadboard for +15 V and wire it to the top red bus strip for +15 V supply.<br />
# Connect the negative terminal of '''CH2''' to a banana terminal on the breadboard for -15 V.<br />
# Connect the positive terminal of '''CH2''' to the black terminal on the breadboard for a ground reference and wire it to the top blue bus strip.<br />
#* Use three jump wires to minimize the parasitic voltage drop due to the breadboard tie points.<br />
# Connect the +15 V bus strip to the second vertical, red bus strip and wire this bus strip to the top of your 15 k&Omega; resistor.<br />
# Turn on the output of the power supply and measure the voltage across the RTD using the DMM.<br />
# '''What is the measured voltage of the RTD at room temperature and how does it compare to your predictions above?'''<br />
# '''What is the temperature, based on your measured voltage and how does it compare with the clock on the wall (or some better measure)?'''<br />
# '''How is the temperature calculation affected by a 1% change in the 15 V input voltage?'''<br />
<br />
====Photodiode amplifier circuit====<br />
<br />
The current produced by the photodiode detector is a few tens of nanoamperes. This very small current must be amplified to a level where it can be easily measured. The data acquisition system you will use can measure voltages between + and -10 V. Use the two-stage op amp circuit shown below to convert the photodiode current to a voltage.<br />
<br />
<center>[[Image:Part1_TransimpedanceAmplifier.jpg|700 px|thumb|center|Multi-stage photodiode amplification circuit]]</center><br />
<br />
You will have to decide how to distribute the gain between the two op amp stages in the circuit. It is best to put as much gain in early stages of a multi-stage amplifier as possible. However, when the gain of a single stage becomes very high, nonidealities of the op amp become bothersome. The first stage of the amplifier will behave reasonably well as long as the gain is less than about 10<sup>6</sup> &Omega;.<br />
<br />
# '''Analyze the photodiode amplifier circuit. Choose resistor values to achieve an overall gain that will yield an output of about 5 V when the input is 500 nA.'''<br />
# Construct the circuit on your breadboard.<br />
#* Disable the power supply before you build.<br />
#* Do not yet connect the photodiode.<br />
#* Orient the LF411 op-amps so that pin 1, which is marked with a dot on the package, is at the upper left.<br />
# Connect the +/- 15 V bus strips to power the op-amps power pins.<br />
# Connect the vertical blue bus strips to the horizontal blue bus strip.<br />
<br />
=====Test connectivity and basic operation=====<br />
<br />
# Verify that all your component connections are correct and match the design.<br />
# Check that wires and components are properly seated in the breadboard and not suspiciously loose.<br />
#* You can use the ohm-meter function of the digital multi-meter to verify continuity.<br />
# Turn on the power supply and verify that +15V appears on pin 7 and -15V on pin 4.<br />
# '''Disconnect the photodiode connection to pin 2 (the <math>v^-</math> input to the op-amp) and measure the output voltages of IC1 and IC2.'''<br />
#* With the photodiode disconnected, the current signal is zero. If you see a significant offset voltage (from 50 to 100 mV) at IC1 pin 6, then you may wish to add (or adjust the value of) <math>R_6</math>.<br />
#* Refer to [http://measure.mit.edu/~20.309/wiki/index.php?title=Real_electronics#Real_op-amps Real Electronics, real op-amps] or ask an instructor to understand and address this non-ideal behavior.<br />
<br />
==Data acquisition hardware subsystem==<br />
<br />
{{:20.309:DAQ System}}<br />
<br />
====Summary of DAQ inputs/outputs====<br />
<br />
A special connector has been prepared for you, connected as shown below. The connections and cable wire colors are also summarized [http://dl.dropbox.com/u/12957607/DAQ_Cable_Assembly_for_DNA_melting_lab.pdf in a memo].<br />
<br />
{| class="wikitable"<br />
|-<br />
! Signal Name<br />
! Signal Location<br />
! Ground Location<br />
! Pin wire color<br />
|-<br />
! DAQ Inputs <br />
|-<br />
! RTD<br />
| AI0+<br />
| AI0-<br />
| +Orange / -Black [lone pair, not with third (red) wire]<br />
|-<br />
! Photodiode <br />
| AI1+ <br />
| AI1-<br />
| +Green / -White<br />
|}<br />
<br />
<center>[[Image:DAQ_Wire_Colors.png|450 px|thumb|center|DAQ Connection Cable]]</center><br />
<br />
==DNAMelter software==<br />
<br />
[[Image:BasicDNAMelterIcon.png|75 px|left]]<br />
Use the <code>Basic DNA Melter GUI</code> program (located on the lab computer desktop) to collect data from the apparatus.<br />
<br />
At the end of an experimental run, use the "Save" button to write the date to a file. The data will be tab-delimited and can be read into Matlab with the <code>load</code> command.<br />
<br />
Documentation for the DNAMelter software is available here: [[DNA Melting: Using the Basic DNAMelter GUI]]<br />
<br />
====If you need to debug the DAQ (skip otherwise!)====<br />
* When debugging problems, it is frequently useful to ascertain whether the data acquisition system is working properly. National Instruments provides an application for this purpose called Measurement and Automation Explorer. Use Measurement and Automation Explorer to verify that the DAQ is properly connected to and communicating with the computer. The program also provides the ability to manually control all of the DAQ system's inputs and outputs.<br />
** Launch Measurement and Automation Explorer by selecting Start->All Applications->National Instruments->Measurement and Automation.<br />
** In the "Configuration" tab, select "Devices and Interfaces" and then "NI-DAQMx Devices." Select "Dev1."<br />
** If Dev 1 does not show up, the problem is that computer cannot communicate with the DAQ system. Ensure that the USB cable is connected to the DAQ system. If the cable is connected and the device still does not show up, unplug the USB cable, count to five, and plug it back in. <br />
** If the DAQ shows up under a different name, disconnect the USB cable. Delete all the "NI USB-6xxx" entries and then reconnect the USB cable. Verify that one entry reappears and that it is called "Dev1." <br />
** Select the "Test Panels" tab to manually control or read signals from the DAQ.<br />
* If you run DNAMelter and there is a data acquisition error, for example, "<code>Data acquisition cannot start!</code>", open Measurement and Automation Explorer and follow the steps above. Run DNAMelter again. If it still doesn't work, grab a beer or ask an instructor for help.<br />
* The DAQ has only one analog-to-digital converter (ADC). A multiplexer circuit sequentially samples voltages from the analog inputs configured in the control software. If any of the inputs is outside the allowable range of +/-10V, all of the inputs may behave erratically. The voltages reported may look reasonable so this can be very misleading. If none of the DAQ readings make sense, ensure that all of the analog voltages are in the allowable range and reset the DAQ by cycling the power of the DAQ (use the switch on the 6341 or disconnect the USB of the 6212).<br />
* If the DAQ gets short-circuited, it may crash. If this happens, Matlab will frequently give a deceptive error message, for example claiming that the limits on the band-pass filter do not satisfy some condition. In this case, correct the connection and cycle the DAQ power.<br />
<br />
==Part 1 measurements==<br />
<br />
Please read this entire section before beginning. Then start at Testing the instrument, with reference back to the sample sections as-needed.<br />
<br />
===Making samples===<br />
<br />
{{Template:Biohazard warning|message=LC Green in DMSO is readily absorbed through skin. Synthetic oligonucleotides may be harmful by inhalation, ingestion, or skin absorption. Wear gloves when handling samples. Wear safety goggles at all times when pipetting the LC Green/DNA samples. Do not create aerosols. The health effects of LC Green have not been thoroughly investigated. See the LC Green and synthetic oligonucleotide under <code>../EHS Guidelines/MSDS Repository</code> in the course locker for more information.}}<br />
<br />
====Sample prep steps====<br />
<br />
#Pipet 500 &mu;L of DNA plus dye solution into a glass vial. <br />
#Pipet up to 200 &mu;L of mineral oil on top of the sample to help prevent evaporation. It will help to pipette into the corners first. Be careful not to get oil on the cuvette sides far above the sample. It will run down during your experiment and cause shifts in the photodiode signal. Place the cap loosely on the vial. <br />
#*Keep the sample vertical to make sure the oil stays on top. <br />
<br />
* You can use the same sample for several heating/cooling cycles.<br />
* Only discard a sample if you lose significant volume due to evaporation or if your signal gets too low. <br />
<br />
To clean your glass vial between samples, flush with alcohol in the waste container and rinse with water at the drain. Suck out residual liquid with the vacuum and drawn Pasteur pipette to the left of the sink.<br />
<br />
====Sample disposal====<br />
<br />
{{Template:Environmental Warning|message=Discard pipette tips with DNA sample residue in the pipette tips or the ''Biohazard Sharps'' container. Do not pour synthetic oligonucleotides with LC Green down the drain. Pour your used samples into the waste container provided in the middle of the wet bench.}}<br />
<br />
===Testing the instrument===<br />
<br />
Once your apparatus is built, use samples of fluorescein (~ 30&mu;M concentration) as a stand-in for LC Green + DNA to test the instrument. Fluorescein is cheap, non-toxic and will not bleach as quickly as LC Green.<br />
<br />
====Making a simple measurement of your instrument's low frequency SNR====<br />
<br />
Carry out a simple estimate of the signal to noise ratio (SNR) of the instrument by comparing the difference between the average signal with a sample present and with water as a null reference. This process requires a vial containing 500 uL of 30 uM fluorescein and a vial containing the same amount of water. <br />
<br />
# Run the <code>Basic DNA Melter GUI</code>. <br />
# Place a vial of DI water in your instrument.<br />
# Clear the data and wait 10 seconds.<br />
# Replace the water vial with the DNA of fluorescein vial, record for another 10 seconds, then save the data. <br />
#* Be sure that all other conditions, such as temperature, are stable throughout the test. <br />
# Segment your data into that when the water was present and that when the fluorescein was present. Calculate the average signal in each case. Record the difference between the two averages as your signal. <br />
# Now discard the water portion of your signal and subtract the average fluorescein signal value from the fluorescein signal.<br />
# Measure the standard deviation of this residual and record as record as your estimate of the noise level.<br />
# '''Calculate the SNR as signal divided by noise and record in your lab report.'''<br />
<br />
===Experiment steps===<br />
<br />
Once the instrument is confirmed to be functioning properly, follow these instructions to generate melting curve data for the 20 bp DNA-LC Green solution.<br />
<br />
#Open and run the <code>Basic DNA Melter GUI</code> in Matlab and follow the instructions there.<br />
# Confirm that block temperature is near room temperature when the Diablotek supply is off.<br />
# Apply heat to the block by switching the Diablotek on to increase the block temperature to 95&deg;C, toggle the Diablotek power in order to hold the temperature long enough for the fluorescence curve to reach a minimum, then allow the block to cool down by switching off the Diablotek.<br />
#* Other approaches may be used. One goal would be to heat the block more slowly, possibly by toggling the current while heating.<br />
# Now prepare a sample using the stock of "beater" DNA.<br />
# Place the vial in the instrument, but keep the LED off until ready to measure the melting curve.<br />
# Measure melting curves of the beater DNA sample as-desired to understand instrument operation.<br />
# Prepare a sample using fresh 20 bp 100 mM KCl DNA.<br />
# Measure a melting curve of the fresh sample.<br />
#* Be sure to save a copy in a local directory.<br />
<br />
{{:DNA Melting Report Requirements for Part 1}}<br />
<br />
==Lab manual sections==<br />
<br />
*[[Lab Manual:Measuring DNA Melting Curves]]<br />
*[[DNA Melting: Simulating DNA Melting - Basics]]<br />
*[[DNA Melting Part 1: Measuring Temperature and Fluorescence]]<br />
*[[DNA Melting Report Requirements for Part 1]]<br />
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]<br />
*[[DNA Melting Report Requirements for Part 2]]<br />
<br />
==References==<br />
<references/><br />
<br />
==Subset of datasheets==<br />
(Many more can be found online or on the course share)<br />
#[http://www.ni.com/pdf/manuals/371931f.pdf National Instruments USB-6212 user manual]<br />
#[http://www.ni.com/pdf/manuals/370784d.pdf National Instruments USB-6341 user manual]<br />
#[http://www.national.com/ds/LF/LF411.pdf Op-amp datasheet]<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/DNA_Melting_Part_1:_Measuring_Temperature_and_FluorescenceDNA Melting Part 1: Measuring Temperature and Fluorescence2015-11-08T21:33:46Z<p>Steven Nagle: /* Test connectivity and basic operation */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
<br />
[[Image:DNA Melting Block Diagram.jpg|frame|DNA melting apparatus block diagram showing the five functional groups of the instrument.]]<br />
<br />
<br />
''In theory, there is no difference between theory and practice. But, in practice, there is.'' <br />
<br />
''&mdash;[http://en.wikiquote.org/wiki/Jan_L._A._van_de_Snepscheut Jan L. A. van de Snepscheut]/[http://en.wikiquote.org/wiki/Yogi_Berra Yogi Berra]/and or [http://c2.com/cgi/wiki?DifferenceBetweenTheoryAndPractice Chuck Reid]'' <br />
<br />
You will construct a basic DNA melting instrument consisting of the five functional groups shown in the block diagram:<br />
* excitation<br />
* heating<br />
* fluorescent detection<br />
* temperature measurement<br />
* data acquisition and control<br />
<br />
A metal block will hold a sample vial in the excitation light path and transfer heat to the sample. The sample contains a combination of DNA, SYBR Green I and a salt. Recall that SYBR Green I fluoresces when it is bound to double stranded DNA (dsDNA) by a factor of up to 1000x compared to when it is bound to single stranded DNA (ssDNA). Detection of this fluorescence provides a relative measure of the fraction of dsDNA to total DNA, which can be compared to predictions from a thermodynamic model.<br />
<br />
The dye has a peak sensitivity to blue light at 497 nm so a blue LED will be used to illuminate the sample. The dye emits green light with an emission peak at 520 nm. A photodiode will be placed at 90&deg; to the LED source to detect the green light emitted by SYBR Green I. Because LEDs have a fairly broad spectrum, first a moderately narrow band-pass filter will be used to limit the excitation wavelength to between 450 and 490 nm. Then a complimentary long-pass emission filter will be placed between the sample and detector to pass only wavelengths longer than 515 nm. This filter combination, when used with this dye system, eliminates any background signal due to the excitation light.<br />
<br />
The sample block will be heated by a TEC (thermoelectric cooler) and its temperature measured by an embedded RTD (resistance temperature detector). Also, since photodiodes produce a very small amount of current, a high gain amplifier will convert the photodiode current into a measurable voltage. The DAQ (data acquisition card) and PC will digitize the amplified photodiode and RTD signals. A Matlab program is provided to record these fluorescence and temperature signals over time and save the data to a file. The file can then be loaded into Matlab for analysis, or if you prefer, Python or equivalent where the signals will be converted into dsDNA concentration and sample temperature. Finally, the dsDNA sample melting temperature will be estimated from a plot of df/dT vs T (''f'' represents the fraction of total DNA that is in double-stranded form) and from a comparison to the thermodynamic model.<br />
<br />
The functional groups in the block diagram are one of two fundamental frameworks used to describe an instrument. The other sub-divides the instrument into categories that often mirror historical areas of engineering. The former is focused on what will be done, including the overall scientific goals and the physical processes to be measured. The latter is focused on how it will be done, e.g. using optics, electronics or perhaps a practical knowledge of thermal management. A good engineer will constantly be thinking of the instrument in both frameworks. <br />
<br />
==Mechanical and thermal components==<br />
Mechanical support for the heating block, sample and optics will be built on a portable optical breadboard from [http://www.thorlabs.com ThorLabs]. Most of the components in the lab are from this supplier because the selection is large, the quality is quite good and the price reasonable. Although you are free to modify the design as the lab progresses, begin a build from the standard design shown above and demonstrated by the example in the lab. Gather the following Thorlabs components but do not start to assemble.<br />
<br />
* 1 MB1224 optical breadboard<br />
* 2 CL5 clamps (or similar)<br />
* 2 1/4-20 5/8" screws<br />
* 4 1/4-20 1/4" screws<br />
<br />
The thermal subsystem includes all components to control heat flow into and out of the sample and monitor its temperature. Heating is accomplished using thermal electric coolers (TECs) in reverse. The hot side is placed in direct contact with the heating block, which is in direct contact with the sample vial. The TECs will be powered by manually controlling the Diablotek power supply. To avoid contamination of the sample or perturbation of its temperature, the temperature of a resistive temperature detector (RTD) embedded in the heating block will be monitored as a proxy for the sample temperature. In this part of the lab, air convection will cool down the sample when the Diablotek is switched off. The heat sink will help and the fan may be connected to a triple-output power supply to help, but these are included primarily for use in Part 2.<br />
<br />
===Parts===<br />
Gather the following components from the bins on the Center Cabinets, the DNA Melting drawer at the wet bench or on the East Cabinets:<br />
<br />
[[Image:CenterCabinets.jpg|right|250px|thumb|'''Center Cabinets''' The bins for most parts that you need will be set out on top of these cabinets, roughly in the center of the lab.]]<br />
[[Image:WetBench.jpg|right|250px|thumb|'''Wet Bench''' Supplies for our wet bench can be found in the three drawers at the left of the bench, the drawer left of the sink and on the wall behind and to the left as you face the wet bench. The drawer at the right of the wet bench contains various supplies for the DNA Melting instrument.]]<br />
[[Image:EastCabinets.jpg|right|250px|thumb|'''East Cabinets''' The center shelves of the East Cabinets contain the electronic breadboards, breadboard jumper wire, TEC connectors and TEC power supplies. Often these supplies are laid out on top of these cabinets.]]<br />
<br />
* These components are already assembled for you: A metal support bracket, having a fan, heatsink, 2 TEC heaters/coolers, and a heating block attached to it<br />
* 3 wire nuts<br />
* 1 Diablotek 250 W power supply (at your station or on a shelf above your station)<br />
* 1 red/black TEC extension wire assembly (in a box on the ''''Center Cabinets'''')<br />
<br />
===Assembly instructions===<br />
<br />
[[Image:TEC_picture.jpg|150 px|thumb|right|TEC showing hot side up when current flows into the red lead]]<br />
<br />
Ensure that the following have been done/attached to your metal bracket:<br />
# The bracket, TECs, fan and heatsink should already be assembled for you.<br />
# Verify that the colors of the leads match. If not, flip one TEC.<br />
# Connect the black lead of the top TEC to the red lead of the bottom TEC using a wire nut.<br />
# Connect the TEC to the extension wire assembly using a wire nut to join the white wire to the red lead of the top TEC and the black wire to the black lead on the bottom TEC.<br />
# Join the white connector on the assembly to the black 2x2 connector of the Diabloteck.<br />
# Test the TECs by holding the them between thumb and finger while briefly flipping the switch on the Diablotek.<br />
#* One side should immediately heat up while the other immediately cools.<br />
#* The top side should be the hot side when the red wires extend down and to the '''left'''.<br />
#* If the Diablotek immediately shuts off (the fan will stop spinning) then there is a short. Check all connections.<br />
# Once the TEC assembly checks out, butter the bottom side of the assembly and place that side on the heat sink with the leads facing to the right.<br />
# Finally, apply thermal grease to the top side of the TEC assembly, place the heating block on top and anchor to the heat sink using the 1/2" long 4-40 screws and plastic washers.<br />
<br />
==Optical subsystem==<br />
<br />
A good optical subsystem design will be compact, stable and simple. It includes the optical components of the excitation and fluorescence detection functional groups from the block diagram. A good design will efficiently illuminate the sample and collect the maximum possible amount of emitted fluorescent light. There are a variety of ways to construct the apparatus. Use the Instructor instrument(s), and the instructions below, as a guide. The set of instructions can help if you're not clear on how to replicate the example system in the lab. In addition to the following parts, you might want to add a few lenses to collimate your LED onto your sample, and to focus the light coming from your sample onto your photodiode. <br />
<br />
* 2 PH2 post holders<br />
* 2 BA1 post holder bases<br />
* 2 1/4-20 1/4" screws<br />
* 2 1/4-20 5/8" screws<br />
* 2 1/4" washers<br />
* 4 CP02 cage plates<br />
* 2 8-32 1/2" setscrews<br />
* 2 TR2 posts<br />
* 6 ER3 cage rails<br />
* 12 4-40 1/4" setscrews (or similar length)<br />
* 4 SML05 lens tubes<br />
* 4 SM1LTRR retaining rings<br />
* 1 blue LED PCB<br />
* 1 D470/40 excitation filter<br />
* 1 E515LPv2 emission filter<br />
* 1 SM1A6 adapter between SM1 and SM05 threads<br />
* 1 SM05PD1A mounted photodiode<br />
<br />
===Assembly instructions===<br />
Use an Instructor instrument or the Solid Works model above as guide to assemble your optical subassembly. Note also that these instructions are also a guid. If you wish to build your own personal design, please do.<br />
<br />
# Attach each PH2 to a BA1 using 1/4" screws.<br />
# Attach two of the CP02 cage plates to two TR2 posts using an 8-32 setscrew.<br />
# Insert the TR2/CP02 assembly into the PH2/BA1 assemblies and lock in place with the TS25H setscrews.<br />
# Insert three ER4 cage rails into each CP02 above and lock in place with SS4S025 set screws or similar.<br />
#* Leave the blank hole on the bottom of the assembly.<br />
# Attach one of the remaining CP02 cage plates on the free end of each assembly above and lock in place.<br />
# Attach each of the above assemblies to the MB1224 using a washer and 1/4-20 screw less than 1" in length.<br />
# Arrange on the MB1224 and adjust CP02 heights as desired.<br />
# Mount the LED PCB in an SML05 lens tube and fix with a retaining ring.<br />
# Mount the excitation and emission filters in each of two lens tubes and fix with retaining ring.<br />
#* The arrow label on the thickness of the filter points ''toward the light source''.<br />
# Thread the SM1A6 adapter into a lens tube and fix with a retaining ring.<br />
# Thread the photodiode into the SM1A6 adapter.<br />
#* Teflon pipe thread tape can be used to hold the photodiode in the desired position.<br />
# Mount each lens tube in the appropriate CP02 cage plates.<br />
# Arrange each the resultant assemblies on the breadboard to most fully illuminate the sample vial and to gather the most of the resultant light on the photodiode.<br />
<br />
==Electronics subsystem==<br />
<br />
The electrical subsystem includes the LED with a current-limiting resistor, the photodiode, the transimpedance amplifier and a temperature detection circuit. The remainder of this section provides an example of each of these systems and instructions to build and test each one. Nevertheless, alternate ideas are welcome. Feel free to discuss with the Instructors.<br />
<br />
===Parts===<br />
<br />
Gather the following components from the electronics drawer to the right of the optics drawers, from the counter top opposite the wet bench and from the cable organizer on the East wall to the right of the white board.<br />
* 1 electronic breadboard<br />
* 1 jump wire kit<br />
* 2 red 3' banana cable or similar<br />
* 1 black 3' banana cable or similar<br />
* individual wire, as-needed from the scrap wire box or the wire spools opposite the wet bench<br />
* 1 62 &Omega; resistor from the center counter top on the South wall<br />
* 1 15 k&Omega; resistor<br />
* other resistors, as-needed for the amplifier<br />
* 1 blue LED from the DNA Melting drawer at the far right of the wet bench<br />
* 2 LF411 op-amps from the electronics drawer<br />
* 4 capacitors between 0.1 and 22 uF from either the electronics drawer or the counter top where the screws are stored.<br />
<br />
===Assembly instructions===<br />
<br />
====LED circuit====<br />
The led circuit consists of a 62 &Omega; resistor in series with a blue, jumbo LED. The circuit will be powered by the 5 volt fixed output of the lab power supply. The 62 &Omega; resistor prevents excessive current from flowing through the LED. Excessive current will let the smoke out of the LED.<br />
<br />
# Build the LED circuit on your electronic breadboard.<br />
#* Don't forget to connect the resistor. Directly connecting the LED to 5 V will blow out the LED.<br />
# Disable the output of the lab power supply.<br />
# Connect the LED circuit to the fixed 5 V output.<br />
<br />
====Temperature detection circuit====<br />
A platinum RTD is embedded inside the heating block. The resistance of the RTD varies with temperature according to the equation ''R<sub>RTD</sub>''=1000 &Omega; + 3.85 ''&theta;'', where ''&theta;'' is the temperature of the RTD in degrees Celsius. The easiest way to make a temperature measurement is to connect the RTD in series with a fixed resistor and a power supply. The output voltage of the resulting divider circuit varies as a function of temperature. <br />
<br />
The maximum current of the RTD is 1 mA. You will use a 15 V supply to the circuit, so the resistor must be at least 14 k&Omega;. Use a 15 k&Omega; resistor.<br />
<br />
# '''Find an equation for ''V<sub>out</sub>'' as a function of temperature. What voltage do you expect given an input voltage of 15 V with the RTD at room temperature.'''<br />
# Turn off the output of the triple-output power supply.<br />
# Connect the RTD to the electronic breadboard.<br />
# '''Measure the resistance of the 15 k&Omega; resistor with a DMM and record the value in your lab report.'''<br />
# Connect the 15k&Omega; resistor in series with the RTD.<br />
# Set the power supply for '''SERIES''' operation.<br />
# Set the output voltage to 15 V on the supply and make note of its exact value using the DMM.<br />
# Set the current limit to 0.1 A.<br />
# Connect the positive terminal of '''CH1''' to a banana terminal on the breadboard for +15 V and wire it to the top red bus strip for +15 V supply.<br />
# Connect the negative terminal of '''CH2''' to a banana terminal on the breadboard for -15 V.<br />
# Connect the positive terminal of '''CH2''' to the black terminal on the breadboard for a ground reference and wire it to the top blue bus strip.<br />
#* Use three jump wires to minimize the parasitic voltage drop due to the breadboard tie points.<br />
# Connect the +15 V bus strip to the second vertical, red bus strip and wire this bus strip to the top of your 15 k&Omega; resistor.<br />
# Turn on the output of the power supply and measure the voltage across the RTD using the DMM.<br />
# '''What is the measured voltage of the RTD at room temperature and how does it compare to your predictions above?'''<br />
# '''What is the temperature, based on your measured voltage and how does it compare with the clock on the wall (or some better measure)?'''<br />
# '''How is the temperature calculation affected by a 1% change in the 15 V input voltage?'''<br />
<br />
====Photodiode amplifier circuit====<br />
<br />
The current produced by the photodiode detector is a few tens of nanoamperes. This very small current must be amplified to a level where it can be easily measured. The data acquisition system you will use can measure voltages between + and -10 V. Use the two-stage op amp circuit shown below to convert the photodiode current to a voltage.<br />
<br />
<center>[[Image:Part1_TransimpedanceAmplifier.jpg|700 px|thumb|center|Multi-stage photodiode amplification circuit]]</center><br />
<br />
You will have to decide how to distribute the gain between the two op amp stages in the circuit. It is best to put as much gain in early stages of a multi-stage amplifier as possible. However, when the gain of a single stage becomes very high, nonidealities of the op amp become bothersome. The first stage of the amplifier will behave reasonably well as long as the gain is less than about 10<sup>6</sup> &Omega;.<br />
<br />
# '''Analyze the photodiode amplifier circuit. Choose resistor values to achieve an overall gain that will yield an output of about 5 V when the input is 500 nA.'''<br />
# Construct the circuit on your breadboard.<br />
#* Disable the power supply before you build.<br />
#* Do not yet connect the photodiode.<br />
#* Orient the LF411 op-amps so that pin 1, which is marked with a dot on the package, is at the upper left.<br />
# Connect the +/- 15 V bus strips to power the op-amps power pins.<br />
# Connect the vertical blue bus strips to the horizontal blue bus strip.<br />
<br />
=====Test connectivity and basic operation=====<br />
<br />
# Verify that all your component connections are correct and match the design.<br />
# Check that wires and components are properly seated in the breadboard and not suspiciously loose.<br />
#* You can use the ohm-meter function of the digital multi-meter to verify continuity.<br />
# Turn on the power supply and verify that +15V appears on pin 7 and -15V on pin 4.<br />
# '''Disconnect the photodiode connection to pin 2 (the input to the op-amp) and measure the output voltages of IC1 and IC2.'''<br />
#* With the photodiode disconnected, the current signal is zero. If you see a significant offset voltage (from 50 to 100 mV) at IC1 pin 6, then you may wish to add (or adjust the value of) <math>R_6</math>.<br />
#* Refer to [http://measure.mit.edu/~20.309/wiki/index.php?title=Real_electronics#Real_op-amps Real Electronics, real op-amps] or ask an instructor to understand and address this non-ideal behavior.<br />
<br />
==Data acquisition hardware subsystem==<br />
<br />
{{:20.309:DAQ System}}<br />
<br />
====Summary of DAQ inputs/outputs====<br />
<br />
A special connector has been prepared for you, connected as shown below. The connections and cable wire colors are also summarized [http://dl.dropbox.com/u/12957607/DAQ_Cable_Assembly_for_DNA_melting_lab.pdf in a memo].<br />
<br />
{| class="wikitable"<br />
|-<br />
! Signal Name<br />
! Signal Location<br />
! Ground Location<br />
! Pin wire color<br />
|-<br />
! DAQ Inputs <br />
|-<br />
! RTD<br />
| AI0+<br />
| AI0-<br />
| +Orange / -Black [lone pair, not with third (red) wire]<br />
|-<br />
! Photodiode <br />
| AI1+ <br />
| AI1-<br />
| +Green / -White<br />
|}<br />
<br />
<center>[[Image:DAQ_Wire_Colors.png|450 px|thumb|center|DAQ Connection Cable]]</center><br />
<br />
==DNAMelter software==<br />
<br />
[[Image:BasicDNAMelterIcon.png|75 px|left]]<br />
Use the <code>Basic DNA Melter GUI</code> program (located on the lab computer desktop) to collect data from the apparatus.<br />
<br />
At the end of an experimental run, use the "Save" button to write the date to a file. The data will be tab-delimited and can be read into Matlab with the <code>load</code> command.<br />
<br />
Documentation for the DNAMelter software is available here: [[DNA Melting: Using the Basic DNAMelter GUI]]<br />
<br />
====If you need to debug the DAQ (skip otherwise!)====<br />
* When debugging problems, it is frequently useful to ascertain whether the data acquisition system is working properly. National Instruments provides an application for this purpose called Measurement and Automation Explorer. Use Measurement and Automation Explorer to verify that the DAQ is properly connected to and communicating with the computer. The program also provides the ability to manually control all of the DAQ system's inputs and outputs.<br />
** Launch Measurement and Automation Explorer by selecting Start->All Applications->National Instruments->Measurement and Automation.<br />
** In the "Configuration" tab, select "Devices and Interfaces" and then "NI-DAQMx Devices." Select "Dev1."<br />
** If Dev 1 does not show up, the problem is that computer cannot communicate with the DAQ system. Ensure that the USB cable is connected to the DAQ system. If the cable is connected and the device still does not show up, unplug the USB cable, count to five, and plug it back in. <br />
** If the DAQ shows up under a different name, disconnect the USB cable. Delete all the "NI USB-6xxx" entries and then reconnect the USB cable. Verify that one entry reappears and that it is called "Dev1." <br />
** Select the "Test Panels" tab to manually control or read signals from the DAQ.<br />
* If you run DNAMelter and there is a data acquisition error, for example, "<code>Data acquisition cannot start!</code>", open Measurement and Automation Explorer and follow the steps above. Run DNAMelter again. If it still doesn't work, grab a beer or ask an instructor for help.<br />
* The DAQ has only one analog-to-digital converter (ADC). A multiplexer circuit sequentially samples voltages from the analog inputs configured in the control software. If any of the inputs is outside the allowable range of +/-10V, all of the inputs may behave erratically. The voltages reported may look reasonable so this can be very misleading. If none of the DAQ readings make sense, ensure that all of the analog voltages are in the allowable range and reset the DAQ by cycling the power of the DAQ (use the switch on the 6341 or disconnect the USB of the 6212).<br />
* If the DAQ gets short-circuited, it may crash. If this happens, Matlab will frequently give a deceptive error message, for example claiming that the limits on the band-pass filter do not satisfy some condition. In this case, correct the connection and cycle the DAQ power.<br />
<br />
==Part 1 measurements==<br />
<br />
Please read this entire section before beginning. Then start at Testing the instrument, with reference back to the sample sections as-needed.<br />
<br />
===Making samples===<br />
<br />
{{Template:Biohazard warning|message=LC Green in DMSO is readily absorbed through skin. Synthetic oligonucleotides may be harmful by inhalation, ingestion, or skin absorption. Wear gloves when handling samples. Wear safety goggles at all times when pipetting the LC Green/DNA samples. Do not create aerosols. The health effects of LC Green have not been thoroughly investigated. See the LC Green and synthetic oligonucleotide under <code>../EHS Guidelines/MSDS Repository</code> in the course locker for more information.}}<br />
<br />
====Sample prep steps====<br />
<br />
#Pipet 500 &mu;L of DNA plus dye solution into a glass vial. <br />
#Pipet up to 200 &mu;L of mineral oil on top of the sample to help prevent evaporation. It will help to pipette into the corners first. Be careful not to get oil on the cuvette sides far above the sample. It will run down during your experiment and cause shifts in the photodiode signal. Place the cap loosely on the vial. <br />
#*Keep the sample vertical to make sure the oil stays on top. <br />
<br />
* You can use the same sample for several heating/cooling cycles.<br />
* Only discard a sample if you lose significant volume due to evaporation or if your signal gets too low. <br />
<br />
To clean your glass vial between samples, flush with alcohol in the waste container and rinse with water at the drain. Suck out residual liquid with the vacuum and drawn Pasteur pipette to the left of the sink.<br />
<br />
====Sample disposal====<br />
<br />
{{Template:Environmental Warning|message=Discard pipette tips with DNA sample residue in the pipette tips or the ''Biohazard Sharps'' container. Do not pour synthetic oligonucleotides with LC Green down the drain. Pour your used samples into the waste container provided in the middle of the wet bench.}}<br />
<br />
===Testing the instrument===<br />
<br />
Once your apparatus is built, use samples of fluorescein (~ 30&mu;M concentration) as a stand-in for LC Green + DNA to test the instrument. Fluorescein is cheap, non-toxic and will not bleach as quickly as LC Green.<br />
<br />
====Making a simple measurement of your instrument's low frequency SNR====<br />
<br />
Carry out a simple estimate of the signal to noise ratio (SNR) of the instrument by comparing the difference between the average signal with a sample present and with water as a null reference. This process requires a vial containing 500 uL of 30 uM fluorescein and a vial containing the same amount of water. <br />
<br />
# Run the <code>Basic DNA Melter GUI</code>. <br />
# Place a vial of DI water in your instrument.<br />
# Clear the data and wait 10 seconds.<br />
# Replace the water vial with the DNA of fluorescein vial, record for another 10 seconds, then save the data. <br />
#* Be sure that all other conditions, such as temperature, are stable throughout the test. <br />
# Segment your data into that when the water was present and that when the fluorescein was present. Calculate the average signal in each case. Record the difference between the two averages as your signal. <br />
# Now discard the water portion of your signal and subtract the average fluorescein signal value from the fluorescein signal.<br />
# Measure the standard deviation of this residual and record as record as your estimate of the noise level.<br />
# '''Calculate the SNR as signal divided by noise and record in your lab report.'''<br />
<br />
===Experiment steps===<br />
<br />
Once the instrument is confirmed to be functioning properly, follow these instructions to generate melting curve data for the 20 bp DNA-LC Green solution.<br />
<br />
#Open and run the <code>Basic DNA Melter GUI</code> in Matlab and follow the instructions there.<br />
# Confirm that block temperature is near room temperature when the Diablotek supply is off.<br />
# Apply heat to the block by switching the Diablotek on to increase the block temperature to 95&deg;C, toggle the Diablotek power in order to hold the temperature long enough for the fluorescence curve to reach a minimum, then allow the block to cool down by switching off the Diablotek.<br />
#* Other approaches may be used. One goal would be to heat the block more slowly, possibly by toggling the current while heating.<br />
# Now prepare a sample using the stock of "beater" DNA.<br />
# Place the vial in the instrument, but keep the LED off until ready to measure the melting curve.<br />
# Measure melting curves of the beater DNA sample as-desired to understand instrument operation.<br />
# Prepare a sample using fresh 20 bp 100 mM KCl DNA.<br />
# Measure a melting curve of the fresh sample.<br />
#* Be sure to save a copy in a local directory.<br />
<br />
{{:DNA Melting Report Requirements for Part 1}}<br />
<br />
==Lab manual sections==<br />
<br />
*[[Lab Manual:Measuring DNA Melting Curves]]<br />
*[[DNA Melting: Simulating DNA Melting - Basics]]<br />
*[[DNA Melting Part 1: Measuring Temperature and Fluorescence]]<br />
*[[DNA Melting Report Requirements for Part 1]]<br />
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]<br />
*[[DNA Melting Report Requirements for Part 2]]<br />
<br />
==References==<br />
<references/><br />
<br />
==Subset of datasheets==<br />
(Many more can be found online or on the course share)<br />
#[http://www.ni.com/pdf/manuals/371931f.pdf National Instruments USB-6212 user manual]<br />
#[http://www.ni.com/pdf/manuals/370784d.pdf National Instruments USB-6341 user manual]<br />
#[http://www.national.com/ds/LF/LF411.pdf Op-amp datasheet]<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/DNA_Melting_Part_1:_Measuring_Temperature_and_FluorescenceDNA Melting Part 1: Measuring Temperature and Fluorescence2015-11-08T18:47:52Z<p>Steven Nagle: /* Optical subsystem */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
<br />
[[Image:DNA Melting Block Diagram.jpg|frame|DNA melting apparatus block diagram showing the five functional groups of the instrument.]]<br />
<br />
<br />
''In theory, there is no difference between theory and practice. But, in practice, there is.'' <br />
<br />
''&mdash;[http://en.wikiquote.org/wiki/Jan_L._A._van_de_Snepscheut Jan L. A. van de Snepscheut]/[http://en.wikiquote.org/wiki/Yogi_Berra Yogi Berra]/and or [http://c2.com/cgi/wiki?DifferenceBetweenTheoryAndPractice Chuck Reid]'' <br />
<br />
You will construct a basic DNA melting instrument consisting of the five functional groups shown in the block diagram:<br />
* excitation<br />
* heating<br />
* fluorescent detection<br />
* temperature measurement<br />
* data acquisition and control<br />
<br />
A metal block will hold a sample vial in the excitation light path and transfer heat to the sample. The sample contains a combination of DNA, SYBR Green I and a salt. Recall that SYBR Green I fluoresces when it is bound to double stranded DNA (dsDNA) by a factor of up to 1000x compared to when it is bound to single stranded DNA (ssDNA). Detection of this fluorescence provides a relative measure of the fraction of dsDNA to total DNA, which can be compared to predictions from a thermodynamic model.<br />
<br />
The dye has a peak sensitivity to blue light at 497 nm so a blue LED will be used to illuminate the sample. The dye emits green light with an emission peak at 520 nm. A photodiode will be placed at 90&deg; to the LED source to detect the green light emitted by SYBR Green I. Because LEDs have a fairly broad spectrum, first a moderately narrow band-pass filter will be used to limit the excitation wavelength to between 450 and 490 nm. Then a complimentary long-pass emission filter will be placed between the sample and detector to pass only wavelengths longer than 515 nm. This filter combination, when used with this dye system, eliminates any background signal due to the excitation light.<br />
<br />
The sample block will be heated by a TEC (thermoelectric cooler) and its temperature measured by an embedded RTD (resistance temperature detector). Also, since photodiodes produce a very small amount of current, a high gain amplifier will convert the photodiode current into a measurable voltage. The DAQ (data acquisition card) and PC will digitize the amplified photodiode and RTD signals. A Matlab program is provided to record these fluorescence and temperature signals over time and save the data to a file. The file can then be loaded into Matlab for analysis, or if you prefer, Python or equivalent where the signals will be converted into dsDNA concentration and sample temperature. Finally, the dsDNA sample melting temperature will be estimated from a plot of df/dT vs T (''f'' represents the fraction of total DNA that is in double-stranded form) and from a comparison to the thermodynamic model.<br />
<br />
The functional groups in the block diagram are one of two fundamental frameworks used to describe an instrument. The other sub-divides the instrument into categories that often mirror historical areas of engineering. The former is focused on what will be done, including the overall scientific goals and the physical processes to be measured. The latter is focused on how it will be done, e.g. using optics, electronics or perhaps a practical knowledge of thermal management. A good engineer will constantly be thinking of the instrument in both frameworks. <br />
<br />
==Mechanical and thermal components==<br />
Mechanical support for the heating block, sample and optics will be built on a portable optical breadboard from [http://www.thorlabs.com ThorLabs]. Most of the components in the lab are from this supplier because the selection is large, the quality is quite good and the price reasonable. Although you are free to modify the design as the lab progresses, begin a build from the standard design shown above and demonstrated by the example in the lab. Gather the following Thorlabs components but do not start to assemble.<br />
<br />
* 1 MB1224 optical breadboard<br />
* 2 CL5 clamps (or similar)<br />
* 2 1/4-20 5/8" screws<br />
* 4 1/4-20 1/4" screws<br />
<br />
The thermal subsystem includes all components to control heat flow into and out of the sample and monitor its temperature. Heating is accomplished using thermal electric coolers (TECs) in reverse. The hot side is placed in direct contact with the heating block, which is in direct contact with the sample vial. The TECs will be powered by manually controlling the Diablotek power supply. To avoid contamination of the sample or perturbation of its temperature, the temperature of a resistive temperature detector (RTD) embedded in the heating block will be monitored as a proxy for the sample temperature. In this part of the lab, air convection will cool down the sample when the Diablotek is switched off. The heat sink will help and the fan may be connected to a triple-output power supply to help, but these are included primarily for use in Part 2.<br />
<br />
===Parts===<br />
Gather the following components from the bins on the Center Cabinets, the DNA Melting drawer at the wet bench or on the East Cabinets:<br />
<br />
[[Image:CenterCabinets.jpg|right|250px|thumb|'''Center Cabinets''' The bins for most parts that you need will be set out on top of these cabinets, roughly in the center of the lab.]]<br />
[[Image:WetBench.jpg|right|250px|thumb|'''Wet Bench''' Supplies for our wet bench can be found in the three drawers at the left of the bench, the drawer left of the sink and on the wall behind and to the left as you face the wet bench. The drawer at the right of the wet bench contains various supplies for the DNA Melting instrument.]]<br />
[[Image:EastCabinets.jpg|right|250px|thumb|'''East Cabinets''' The center shelves of the East Cabinets contain the electronic breadboards, breadboard jumper wire, TEC connectors and TEC power supplies. Often these supplies are laid out on top of these cabinets.]]<br />
<br />
* These components are already assembled for you: A metal support bracket, having a fan, heatsink, 2 TEC heaters/coolers, and a heating block attached to it<br />
* 3 wire nuts<br />
* 1 Diablotek 250 W power supply (at your station or on a shelf above your station)<br />
* 1 red/black TEC extension wire assembly (in a box on the ''''Center Cabinets'''')<br />
<br />
===Assembly instructions===<br />
<br />
[[Image:TEC_picture.jpg|150 px|thumb|right|TEC showing hot side up when current flows into the red lead]]<br />
<br />
Ensure that the following have been done/attached to your metal bracket:<br />
# The bracket, TECs, fan and heatsink should already be assembled for you.<br />
# Verify that the colors of the leads match. If not, flip one TEC.<br />
# Connect the black lead of the top TEC to the red lead of the bottom TEC using a wire nut.<br />
# Connect the TEC to the extension wire assembly using a wire nut to join the white wire to the red lead of the top TEC and the black wire to the black lead on the bottom TEC.<br />
# Join the white connector on the assembly to the black 2x2 connector of the Diabloteck.<br />
# Test the TECs by holding the them between thumb and finger while briefly flipping the switch on the Diablotek.<br />
#* One side should immediately heat up while the other immediately cools.<br />
#* The top side should be the hot side when the red wires extend down and to the '''left'''.<br />
#* If the Diablotek immediately shuts off (the fan will stop spinning) then there is a short. Check all connections.<br />
# Once the TEC assembly checks out, butter the bottom side of the assembly and place that side on the heat sink with the leads facing to the right.<br />
# Finally, apply thermal grease to the top side of the TEC assembly, place the heating block on top and anchor to the heat sink using the 1/2" long 4-40 screws and plastic washers.<br />
<br />
==Optical subsystem==<br />
<br />
A good optical subsystem design will be compact, stable and simple. It includes the optical components of the excitation and fluorescence detection functional groups from the block diagram. A good design will efficiently illuminate the sample and collect the maximum possible amount of emitted fluorescent light. There are a variety of ways to construct the apparatus. Use the Instructor instrument(s), and the instructions below, as a guide. The set of instructions can help if you're not clear on how to replicate the example system in the lab. In addition to the following parts, you might want to add a few lenses to collimate your LED onto your sample, and to focus the light coming from your sample onto your photodiode. <br />
<br />
* 2 PH2 post holders<br />
* 2 BA1 post holder bases<br />
* 2 1/4-20 1/4" screws<br />
* 2 1/4-20 5/8" screws<br />
* 2 1/4" washers<br />
* 4 CP02 cage plates<br />
* 2 8-32 1/2" setscrews<br />
* 2 TR2 posts<br />
* 6 ER3 cage rails<br />
* 12 4-40 1/4" setscrews (or similar length)<br />
* 4 SML05 lens tubes<br />
* 4 SM1LTRR retaining rings<br />
* 1 blue LED PCB<br />
* 1 D470/40 excitation filter<br />
* 1 E515LPv2 emission filter<br />
* 1 SM1A6 adapter between SM1 and SM05 threads<br />
* 1 SM05PD1A mounted photodiode<br />
<br />
===Assembly instructions===<br />
Use an Instructor instrument or the Solid Works model above as guide to assemble your optical subassembly. Note also that these instructions are also a guid. If you wish to build your own personal design, please do.<br />
<br />
# Attach each PH2 to a BA1 using 1/4" screws.<br />
# Attach two of the CP02 cage plates to two TR2 posts using an 8-32 setscrew.<br />
# Insert the TR2/CP02 assembly into the PH2/BA1 assemblies and lock in place with the TS25H setscrews.<br />
# Insert three ER4 cage rails into each CP02 above and lock in place with SS4S025 set screws or similar.<br />
#* Leave the blank hole on the bottom of the assembly.<br />
# Attach one of the remaining CP02 cage plates on the free end of each assembly above and lock in place.<br />
# Attach each of the above assemblies to the MB1224 using a washer and 1/4-20 screw less than 1" in length.<br />
# Arrange on the MB1224 and adjust CP02 heights as desired.<br />
# Mount the LED PCB in an SML05 lens tube and fix with a retaining ring.<br />
# Mount the excitation and emission filters in each of two lens tubes and fix with retaining ring.<br />
#* The arrow label on the thickness of the filter points ''toward the light source''.<br />
# Thread the SM1A6 adapter into a lens tube and fix with a retaining ring.<br />
# Thread the photodiode into the SM1A6 adapter.<br />
#* Teflon pipe thread tape can be used to hold the photodiode in the desired position.<br />
# Mount each lens tube in the appropriate CP02 cage plates.<br />
# Arrange each the resultant assemblies on the breadboard to most fully illuminate the sample vial and to gather the most of the resultant light on the photodiode.<br />
<br />
==Electronics subsystem==<br />
<br />
The electrical subsystem includes the LED with a current-limiting resistor, the photodiode, the transimpedance amplifier and a temperature detection circuit. The remainder of this section provides an example of each of these systems and instructions to build and test each one. Nevertheless, alternate ideas are welcome. Feel free to discuss with the Instructors.<br />
<br />
===Parts===<br />
<br />
Gather the following components from the electronics drawer to the right of the optics drawers, from the counter top opposite the wet bench and from the cable organizer on the East wall to the right of the white board.<br />
* 1 electronic breadboard<br />
* 1 jump wire kit<br />
* 2 red 3' banana cable or similar<br />
* 1 black 3' banana cable or similar<br />
* individual wire, as-needed from the scrap wire box or the wire spools opposite the wet bench<br />
* 1 62 &Omega; resistor from the center counter top on the South wall<br />
* 1 15 k&Omega; resistor<br />
* other resistors, as-needed for the amplifier<br />
* 1 blue LED from the DNA Melting drawer at the far right of the wet bench<br />
* 2 LF411 op-amps from the electronics drawer<br />
* 4 capacitors between 0.1 and 22 uF from either the electronics drawer or the counter top where the screws are stored.<br />
<br />
===Assembly instructions===<br />
<br />
====LED circuit====<br />
The led circuit consists of a 62 &Omega; resistor in series with a blue, jumbo LED. The circuit will be powered by the 5 volt fixed output of the lab power supply. The 62 &Omega; resistor prevents excessive current from flowing through the LED. Excessive current will let the smoke out of the LED.<br />
<br />
# Build the LED circuit on your electronic breadboard.<br />
#* Don't forget to connect the resistor. Directly connecting the LED to 5 V will blow out the LED.<br />
# Disable the output of the lab power supply.<br />
# Connect the LED circuit to the fixed 5 V output.<br />
<br />
====Temperature detection circuit====<br />
A platinum RTD is embedded inside the heating block. The resistance of the RTD varies with temperature according to the equation ''R<sub>RTD</sub>''=1000 &Omega; + 3.85 ''&theta;'', where ''&theta;'' is the temperature of the RTD in degrees Celsius. The easiest way to make a temperature measurement is to connect the RTD in series with a fixed resistor and a power supply. The output voltage of the resulting divider circuit varies as a function of temperature. <br />
<br />
The maximum current of the RTD is 1 mA. You will use a 15 V supply to the circuit, so the resistor must be at least 14 k&Omega;. Use a 15 k&Omega; resistor.<br />
<br />
# '''Find an equation for ''V<sub>out</sub>'' as a function of temperature. What voltage do you expect given an input voltage of 15 V with the RTD at room temperature.'''<br />
# Turn off the output of the triple-output power supply.<br />
# Connect the RTD to the electronic breadboard.<br />
# '''Measure the resistance of the 15 k&Omega; resistor with a DMM and record the value in your lab report.'''<br />
# Connect the 15k&Omega; resistor in series with the RTD.<br />
# Set the power supply for '''SERIES''' operation.<br />
# Set the output voltage to 15 V on the supply and make note of its exact value using the DMM.<br />
# Set the current limit to 0.1 A.<br />
# Connect the positive terminal of '''CH1''' to a banana terminal on the breadboard for +15 V and wire it to the top red bus strip for +15 V supply.<br />
# Connect the negative terminal of '''CH2''' to a banana terminal on the breadboard for -15 V.<br />
# Connect the positive terminal of '''CH2''' to the black terminal on the breadboard for a ground reference and wire it to the top blue bus strip.<br />
#* Use three jump wires to minimize the parasitic voltage drop due to the breadboard tie points.<br />
# Connect the +15 V bus strip to the second vertical, red bus strip and wire this bus strip to the top of your 15 k&Omega; resistor.<br />
# Turn on the output of the power supply and measure the voltage across the RTD using the DMM.<br />
# '''What is the measured voltage of the RTD at room temperature and how does it compare to your predictions above?'''<br />
# '''What is the temperature, based on your measured voltage and how does it compare with the clock on the wall (or some better measure)?'''<br />
# '''How is the temperature calculation affected by a 1% change in the 15 V input voltage?'''<br />
<br />
====Photodiode amplifier circuit====<br />
<br />
The current produced by the photodiode detector is a few tens of nanoamperes. This very small current must be amplified to a level where it can be easily measured. The data acquisition system you will use can measure voltages between + and -10 V. Use the two-stage op amp circuit shown below to convert the photodiode current to a voltage.<br />
<br />
<center>[[Image:Part1_TransimpedanceAmplifier.jpg|700 px|thumb|center|Multi-stage photodiode amplification circuit]]</center><br />
<br />
You will have to decide how to distribute the gain between the two op amp stages in the circuit. It is best to put as much gain in early stages of a multi-stage amplifier as possible. However, when the gain of a single stage becomes very high, nonidealities of the op amp become bothersome. The first stage of the amplifier will behave reasonably well as long as the gain is less than about 10<sup>6</sup> &Omega;.<br />
<br />
# '''Analyze the photodiode amplifier circuit. Choose resistor values to achieve an overall gain that will yield an output of about 5 V when the input is 500 nA.'''<br />
# Construct the circuit on your breadboard.<br />
#* Disable the power supply before you build.<br />
#* Do not yet connect the photodiode.<br />
#* Orient the LF411 op-amps so that pin 1, which is marked with a dot on the package, is at the upper left.<br />
# Connect the +/- 15 V bus strips to power the op-amps power pins.<br />
# Connect the vertical blue bus strips to the horizontal blue bus strip.<br />
<br />
=====Test connectivity and basic operation=====<br />
<br />
# Verify that all your component connections are correct and match the design.<br />
# Check that wires and components are properly seated in the breadboard and not suspiciously loose.<br />
#* You can use the ohm-meter function of the digital multi-meter to verify continuity.<br />
# Turn on the power supply and verify that +15V appears on pin 7 and -15V on pin 4.<br />
# '''Short pins 2 and 3 (the V- and V+ inputs to the op-amp) with a small wire and measure the output voltages of IC1 and IC2.'''<br />
#* With the photodiode shorted the current signal is zero, but you may still see a significant offset voltage from 50 to 100 mV at IC1 pin 6.<br />
#* Refer to [http://measure.mit.edu/~20.309/wiki/index.php?title=Real_electronics#Real_op-amps Real Electronics, real op-amps] or ask an instructor to understand and address this non-ideal behavior.<br />
<br />
==Data acquisition hardware subsystem==<br />
<br />
{{:20.309:DAQ System}}<br />
<br />
====Summary of DAQ inputs/outputs====<br />
<br />
A special connector has been prepared for you, connected as shown below. The connections and cable wire colors are also summarized [http://dl.dropbox.com/u/12957607/DAQ_Cable_Assembly_for_DNA_melting_lab.pdf in a memo].<br />
<br />
{| class="wikitable"<br />
|-<br />
! Signal Name<br />
! Signal Location<br />
! Ground Location<br />
! Pin wire color<br />
|-<br />
! DAQ Inputs <br />
|-<br />
! RTD<br />
| AI0+<br />
| AI0-<br />
| +Orange / -Black [lone pair, not with third (red) wire]<br />
|-<br />
! Photodiode <br />
| AI1+ <br />
| AI1-<br />
| +Green / -White<br />
|}<br />
<br />
<center>[[Image:DAQ_Wire_Colors.png|450 px|thumb|center|DAQ Connection Cable]]</center><br />
<br />
==DNAMelter software==<br />
<br />
[[Image:BasicDNAMelterIcon.png|75 px|left]]<br />
Use the <code>Basic DNA Melter GUI</code> program (located on the lab computer desktop) to collect data from the apparatus.<br />
<br />
At the end of an experimental run, use the "Save" button to write the date to a file. The data will be tab-delimited and can be read into Matlab with the <code>load</code> command.<br />
<br />
Documentation for the DNAMelter software is available here: [[DNA Melting: Using the Basic DNAMelter GUI]]<br />
<br />
====If you need to debug the DAQ (skip otherwise!)====<br />
* When debugging problems, it is frequently useful to ascertain whether the data acquisition system is working properly. National Instruments provides an application for this purpose called Measurement and Automation Explorer. Use Measurement and Automation Explorer to verify that the DAQ is properly connected to and communicating with the computer. The program also provides the ability to manually control all of the DAQ system's inputs and outputs.<br />
** Launch Measurement and Automation Explorer by selecting Start->All Applications->National Instruments->Measurement and Automation.<br />
** In the "Configuration" tab, select "Devices and Interfaces" and then "NI-DAQMx Devices." Select "Dev1."<br />
** If Dev 1 does not show up, the problem is that computer cannot communicate with the DAQ system. Ensure that the USB cable is connected to the DAQ system. If the cable is connected and the device still does not show up, unplug the USB cable, count to five, and plug it back in. <br />
** If the DAQ shows up under a different name, disconnect the USB cable. Delete all the "NI USB-6xxx" entries and then reconnect the USB cable. Verify that one entry reappears and that it is called "Dev1." <br />
** Select the "Test Panels" tab to manually control or read signals from the DAQ.<br />
* If you run DNAMelter and there is a data acquisition error, for example, "<code>Data acquisition cannot start!</code>", open Measurement and Automation Explorer and follow the steps above. Run DNAMelter again. If it still doesn't work, grab a beer or ask an instructor for help.<br />
* The DAQ has only one analog-to-digital converter (ADC). A multiplexer circuit sequentially samples voltages from the analog inputs configured in the control software. If any of the inputs is outside the allowable range of +/-10V, all of the inputs may behave erratically. The voltages reported may look reasonable so this can be very misleading. If none of the DAQ readings make sense, ensure that all of the analog voltages are in the allowable range and reset the DAQ by cycling the power of the DAQ (use the switch on the 6341 or disconnect the USB of the 6212).<br />
* If the DAQ gets short-circuited, it may crash. If this happens, Matlab will frequently give a deceptive error message, for example claiming that the limits on the band-pass filter do not satisfy some condition. In this case, correct the connection and cycle the DAQ power.<br />
<br />
==Part 1 measurements==<br />
<br />
Please read this entire section before beginning. Then start at Testing the instrument, with reference back to the sample sections as-needed.<br />
<br />
===Making samples===<br />
<br />
{{Template:Biohazard warning|message=LC Green in DMSO is readily absorbed through skin. Synthetic oligonucleotides may be harmful by inhalation, ingestion, or skin absorption. Wear gloves when handling samples. Wear safety goggles at all times when pipetting the LC Green/DNA samples. Do not create aerosols. The health effects of LC Green have not been thoroughly investigated. See the LC Green and synthetic oligonucleotide under <code>../EHS Guidelines/MSDS Repository</code> in the course locker for more information.}}<br />
<br />
====Sample prep steps====<br />
<br />
#Pipet 500 &mu;L of DNA plus dye solution into a glass vial. <br />
#Pipet up to 200 &mu;L of mineral oil on top of the sample to help prevent evaporation. It will help to pipette into the corners first. Be careful not to get oil on the cuvette sides far above the sample. It will run down during your experiment and cause shifts in the photodiode signal. Place the cap loosely on the vial. <br />
#*Keep the sample vertical to make sure the oil stays on top. <br />
<br />
* You can use the same sample for several heating/cooling cycles.<br />
* Only discard a sample if you lose significant volume due to evaporation or if your signal gets too low. <br />
<br />
To clean your glass vial between samples, flush with alcohol in the waste container and rinse with water at the drain. Suck out residual liquid with the vacuum and drawn Pasteur pipette to the left of the sink.<br />
<br />
====Sample disposal====<br />
<br />
{{Template:Environmental Warning|message=Discard pipette tips with DNA sample residue in the pipette tips or the ''Biohazard Sharps'' container. Do not pour synthetic oligonucleotides with LC Green down the drain. Pour your used samples into the waste container provided in the middle of the wet bench.}}<br />
<br />
===Testing the instrument===<br />
<br />
Once your apparatus is built, use samples of fluorescein (~ 30&mu;M concentration) as a stand-in for LC Green + DNA to test the instrument. Fluorescein is cheap, non-toxic and will not bleach as quickly as LC Green.<br />
<br />
====Making a simple measurement of your instrument's low frequency SNR====<br />
<br />
Carry out a simple estimate of the signal to noise ratio (SNR) of the instrument by comparing the difference between the average signal with a sample present and with water as a null reference. This process requires a vial containing 500 uL of 30 uM fluorescein and a vial containing the same amount of water. <br />
<br />
# Run the <code>Basic DNA Melter GUI</code>. <br />
# Place a vial of DI water in your instrument.<br />
# Clear the data and wait 10 seconds.<br />
# Replace the water vial with the DNA of fluorescein vial, record for another 10 seconds, then save the data. <br />
#* Be sure that all other conditions, such as temperature, are stable throughout the test. <br />
# Segment your data into that when the water was present and that when the fluorescein was present. Calculate the average signal in each case. Record the difference between the two averages as your signal. <br />
# Now discard the water portion of your signal and subtract the average fluorescein signal value from the fluorescein signal.<br />
# Measure the standard deviation of this residual and record as record as your estimate of the noise level.<br />
# '''Calculate the SNR as signal divided by noise and record in your lab report.'''<br />
<br />
===Experiment steps===<br />
<br />
Once the instrument is confirmed to be functioning properly, follow these instructions to generate melting curve data for the 20 bp DNA-LC Green solution.<br />
<br />
#Open and run the <code>Basic DNA Melter GUI</code> in Matlab and follow the instructions there.<br />
# Confirm that block temperature is near room temperature when the Diablotek supply is off.<br />
# Apply heat to the block by switching the Diablotek on to increase the block temperature to 95&deg;C, toggle the Diablotek power in order to hold the temperature long enough for the fluorescence curve to reach a minimum, then allow the block to cool down by switching off the Diablotek.<br />
#* Other approaches may be used. One goal would be to heat the block more slowly, possibly by toggling the current while heating.<br />
# Now prepare a sample using the stock of "beater" DNA.<br />
# Place the vial in the instrument, but keep the LED off until ready to measure the melting curve.<br />
# Measure melting curves of the beater DNA sample as-desired to understand instrument operation.<br />
# Prepare a sample using fresh 20 bp 100 mM KCl DNA.<br />
# Measure a melting curve of the fresh sample.<br />
#* Be sure to save a copy in a local directory.<br />
<br />
{{:DNA Melting Report Requirements for Part 1}}<br />
<br />
==Lab manual sections==<br />
<br />
*[[Lab Manual:Measuring DNA Melting Curves]]<br />
*[[DNA Melting: Simulating DNA Melting - Basics]]<br />
*[[DNA Melting Part 1: Measuring Temperature and Fluorescence]]<br />
*[[DNA Melting Report Requirements for Part 1]]<br />
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]<br />
*[[DNA Melting Report Requirements for Part 2]]<br />
<br />
==References==<br />
<references/><br />
<br />
==Subset of datasheets==<br />
(Many more can be found online or on the course share)<br />
#[http://www.ni.com/pdf/manuals/371931f.pdf National Instruments USB-6212 user manual]<br />
#[http://www.ni.com/pdf/manuals/370784d.pdf National Instruments USB-6341 user manual]<br />
#[http://www.national.com/ds/LF/LF411.pdf Op-amp datasheet]<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/DNA_Melting_Part_1:_Measuring_Temperature_and_FluorescenceDNA Melting Part 1: Measuring Temperature and Fluorescence2015-11-08T18:44:44Z<p>Steven Nagle: /* Mechanical and thermal components */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
<br />
[[Image:DNA Melting Block Diagram.jpg|frame|DNA melting apparatus block diagram showing the five functional groups of the instrument.]]<br />
<br />
<br />
''In theory, there is no difference between theory and practice. But, in practice, there is.'' <br />
<br />
''&mdash;[http://en.wikiquote.org/wiki/Jan_L._A._van_de_Snepscheut Jan L. A. van de Snepscheut]/[http://en.wikiquote.org/wiki/Yogi_Berra Yogi Berra]/and or [http://c2.com/cgi/wiki?DifferenceBetweenTheoryAndPractice Chuck Reid]'' <br />
<br />
You will construct a basic DNA melting instrument consisting of the five functional groups shown in the block diagram:<br />
* excitation<br />
* heating<br />
* fluorescent detection<br />
* temperature measurement<br />
* data acquisition and control<br />
<br />
A metal block will hold a sample vial in the excitation light path and transfer heat to the sample. The sample contains a combination of DNA, SYBR Green I and a salt. Recall that SYBR Green I fluoresces when it is bound to double stranded DNA (dsDNA) by a factor of up to 1000x compared to when it is bound to single stranded DNA (ssDNA). Detection of this fluorescence provides a relative measure of the fraction of dsDNA to total DNA, which can be compared to predictions from a thermodynamic model.<br />
<br />
The dye has a peak sensitivity to blue light at 497 nm so a blue LED will be used to illuminate the sample. The dye emits green light with an emission peak at 520 nm. A photodiode will be placed at 90&deg; to the LED source to detect the green light emitted by SYBR Green I. Because LEDs have a fairly broad spectrum, first a moderately narrow band-pass filter will be used to limit the excitation wavelength to between 450 and 490 nm. Then a complimentary long-pass emission filter will be placed between the sample and detector to pass only wavelengths longer than 515 nm. This filter combination, when used with this dye system, eliminates any background signal due to the excitation light.<br />
<br />
The sample block will be heated by a TEC (thermoelectric cooler) and its temperature measured by an embedded RTD (resistance temperature detector). Also, since photodiodes produce a very small amount of current, a high gain amplifier will convert the photodiode current into a measurable voltage. The DAQ (data acquisition card) and PC will digitize the amplified photodiode and RTD signals. A Matlab program is provided to record these fluorescence and temperature signals over time and save the data to a file. The file can then be loaded into Matlab for analysis, or if you prefer, Python or equivalent where the signals will be converted into dsDNA concentration and sample temperature. Finally, the dsDNA sample melting temperature will be estimated from a plot of df/dT vs T (''f'' represents the fraction of total DNA that is in double-stranded form) and from a comparison to the thermodynamic model.<br />
<br />
The functional groups in the block diagram are one of two fundamental frameworks used to describe an instrument. The other sub-divides the instrument into categories that often mirror historical areas of engineering. The former is focused on what will be done, including the overall scientific goals and the physical processes to be measured. The latter is focused on how it will be done, e.g. using optics, electronics or perhaps a practical knowledge of thermal management. A good engineer will constantly be thinking of the instrument in both frameworks. <br />
<br />
==Mechanical and thermal components==<br />
Mechanical support for the heating block, sample and optics will be built on a portable optical breadboard from [http://www.thorlabs.com ThorLabs]. Most of the components in the lab are from this supplier because the selection is large, the quality is quite good and the price reasonable. Although you are free to modify the design as the lab progresses, begin a build from the standard design shown above and demonstrated by the example in the lab. Gather the following Thorlabs components but do not start to assemble.<br />
<br />
* 1 MB1224 optical breadboard<br />
* 2 CL5 clamps (or similar)<br />
* 2 1/4-20 5/8" screws<br />
* 4 1/4-20 1/4" screws<br />
<br />
The thermal subsystem includes all components to control heat flow into and out of the sample and monitor its temperature. Heating is accomplished using thermal electric coolers (TECs) in reverse. The hot side is placed in direct contact with the heating block, which is in direct contact with the sample vial. The TECs will be powered by manually controlling the Diablotek power supply. To avoid contamination of the sample or perturbation of its temperature, the temperature of a resistive temperature detector (RTD) embedded in the heating block will be monitored as a proxy for the sample temperature. In this part of the lab, air convection will cool down the sample when the Diablotek is switched off. The heat sink will help and the fan may be connected to a triple-output power supply to help, but these are included primarily for use in Part 2.<br />
<br />
===Parts===<br />
Gather the following components from the bins on the Center Cabinets, the DNA Melting drawer at the wet bench or on the East Cabinets:<br />
<br />
[[Image:CenterCabinets.jpg|right|250px|thumb|'''Center Cabinets''' The bins for most parts that you need will be set out on top of these cabinets, roughly in the center of the lab.]]<br />
[[Image:WetBench.jpg|right|250px|thumb|'''Wet Bench''' Supplies for our wet bench can be found in the three drawers at the left of the bench, the drawer left of the sink and on the wall behind and to the left as you face the wet bench. The drawer at the right of the wet bench contains various supplies for the DNA Melting instrument.]]<br />
[[Image:EastCabinets.jpg|right|250px|thumb|'''East Cabinets''' The center shelves of the East Cabinets contain the electronic breadboards, breadboard jumper wire, TEC connectors and TEC power supplies. Often these supplies are laid out on top of these cabinets.]]<br />
<br />
* These components are already assembled for you: A metal support bracket, having a fan, heatsink, 2 TEC heaters/coolers, and a heating block attached to it<br />
* 3 wire nuts<br />
* 1 Diablotek 250 W power supply (at your station or on a shelf above your station)<br />
* 1 red/black TEC extension wire assembly (in a box on the ''''Center Cabinets'''')<br />
<br />
===Assembly instructions===<br />
<br />
[[Image:TEC_picture.jpg|150 px|thumb|right|TEC showing hot side up when current flows into the red lead]]<br />
<br />
Ensure that the following have been done/attached to your metal bracket:<br />
# The bracket, TECs, fan and heatsink should already be assembled for you.<br />
# Verify that the colors of the leads match. If not, flip one TEC.<br />
# Connect the black lead of the top TEC to the red lead of the bottom TEC using a wire nut.<br />
# Connect the TEC to the extension wire assembly using a wire nut to join the white wire to the red lead of the top TEC and the black wire to the black lead on the bottom TEC.<br />
# Join the white connector on the assembly to the black 2x2 connector of the Diabloteck.<br />
# Test the TECs by holding the them between thumb and finger while briefly flipping the switch on the Diablotek.<br />
#* One side should immediately heat up while the other immediately cools.<br />
#* The top side should be the hot side when the red wires extend down and to the '''left'''.<br />
#* If the Diablotek immediately shuts off (the fan will stop spinning) then there is a short. Check all connections.<br />
# Once the TEC assembly checks out, butter the bottom side of the assembly and place that side on the heat sink with the leads facing to the right.<br />
# Finally, apply thermal grease to the top side of the TEC assembly, place the heating block on top and anchor to the heat sink using the 1/2" long 4-40 screws and plastic washers.<br />
<br />
==Optical subsystem==<br />
<br />
A good optical subsystem design will be compact, stable and simple. It includes the optical components of the excitation and fluorescence detection functional groups from the block diagram. A good design will efficiently illuminate the sample and collect the maximum possible amount of emitted fluorescent light. There are a variety of ways to construct the apparatus. Use the model above and the example system in the lab as guides.<br />
<br />
The following is a set of instructions if you're not clear on how to replicate the example system in the lab. In addition to the following parts, you might want to add a few lenses to collimate your LED onto your sample, and to focus the light coming from your sample onto your photodiode. <br />
<br />
* 2 PH2 post holders<br />
* 2 BA1 post holder bases<br />
* 2 1/4-20 1/4" screws<br />
* 2 1/4-20 5/8" screws<br />
* 2 1/4" washers<br />
* 4 CP02 cage plates<br />
* 2 8-32 1/2" setscrews<br />
* 2 TR2 posts<br />
* 6 ER3 cage rails<br />
* 12 4-40 1/4" setscrews (or similar length)<br />
* 4 SML05 lens tubes<br />
* 4 SM1LTRR retaining rings<br />
* 1 blue LED PCB<br />
* 1 D470/40 excitation filter<br />
* 1 E515LPv2 emission filter<br />
* 1 SM1A6 adapter between SM1 and SM05 threads<br />
* 1 SM05PD1A mounted photodiode<br />
<br />
===Assembly instructions===<br />
Use an Instructor instrument or the Solid Works model above as guide to assemble your optical subassembly. Note also that these instructions are also a guid. If you wish to build your own personal design, please do.<br />
<br />
# Attach each PH2 to a BA1 using 1/4" screws.<br />
# Attach two of the CP02 cage plates to two TR2 posts using an 8-32 setscrew.<br />
# Insert the TR2/CP02 assembly into the PH2/BA1 assemblies and lock in place with the TS25H setscrews.<br />
# Insert three ER4 cage rails into each CP02 above and lock in place with SS4S025 set screws or similar.<br />
#* Leave the blank hole on the bottom of the assembly.<br />
# Attach one of the remaining CP02 cage plates on the free end of each assembly above and lock in place.<br />
# Attach each of the above assemblies to the MB1224 using a washer and 1/4-20 screw less than 1" in length.<br />
# Arrange on the MB1224 and adjust CP02 heights as desired.<br />
# Mount the LED PCB in an SML05 lens tube and fix with a retaining ring.<br />
# Mount the excitation and emission filters in each of two lens tubes and fix with retaining ring.<br />
#* The arrow label on the thickness of the filter points ''toward the light source''.<br />
# Thread the SM1A6 adapter into a lens tube and fix with a retaining ring.<br />
# Thread the photodiode into the SM1A6 adapter.<br />
#* Teflon pipe thread tape can be used to hold the photodiode in the desired position.<br />
# Mount each lens tube in the appropriate CP02 cage plates.<br />
# Arrange each the resultant assemblies on the breadboard to most fully illuminate the sample vial and to gather the most of the resultant light on the photodiode.<br />
<br />
==Electronics subsystem==<br />
<br />
The electrical subsystem includes the LED with a current-limiting resistor, the photodiode, the transimpedance amplifier and a temperature detection circuit. The remainder of this section provides an example of each of these systems and instructions to build and test each one. Nevertheless, alternate ideas are welcome. Feel free to discuss with the Instructors.<br />
<br />
===Parts===<br />
<br />
Gather the following components from the electronics drawer to the right of the optics drawers, from the counter top opposite the wet bench and from the cable organizer on the East wall to the right of the white board.<br />
* 1 electronic breadboard<br />
* 1 jump wire kit<br />
* 2 red 3' banana cable or similar<br />
* 1 black 3' banana cable or similar<br />
* individual wire, as-needed from the scrap wire box or the wire spools opposite the wet bench<br />
* 1 62 &Omega; resistor from the center counter top on the South wall<br />
* 1 15 k&Omega; resistor<br />
* other resistors, as-needed for the amplifier<br />
* 1 blue LED from the DNA Melting drawer at the far right of the wet bench<br />
* 2 LF411 op-amps from the electronics drawer<br />
* 4 capacitors between 0.1 and 22 uF from either the electronics drawer or the counter top where the screws are stored.<br />
<br />
===Assembly instructions===<br />
<br />
====LED circuit====<br />
The led circuit consists of a 62 &Omega; resistor in series with a blue, jumbo LED. The circuit will be powered by the 5 volt fixed output of the lab power supply. The 62 &Omega; resistor prevents excessive current from flowing through the LED. Excessive current will let the smoke out of the LED.<br />
<br />
# Build the LED circuit on your electronic breadboard.<br />
#* Don't forget to connect the resistor. Directly connecting the LED to 5 V will blow out the LED.<br />
# Disable the output of the lab power supply.<br />
# Connect the LED circuit to the fixed 5 V output.<br />
<br />
====Temperature detection circuit====<br />
A platinum RTD is embedded inside the heating block. The resistance of the RTD varies with temperature according to the equation ''R<sub>RTD</sub>''=1000 &Omega; + 3.85 ''&theta;'', where ''&theta;'' is the temperature of the RTD in degrees Celsius. The easiest way to make a temperature measurement is to connect the RTD in series with a fixed resistor and a power supply. The output voltage of the resulting divider circuit varies as a function of temperature. <br />
<br />
The maximum current of the RTD is 1 mA. You will use a 15 V supply to the circuit, so the resistor must be at least 14 k&Omega;. Use a 15 k&Omega; resistor.<br />
<br />
# '''Find an equation for ''V<sub>out</sub>'' as a function of temperature. What voltage do you expect given an input voltage of 15 V with the RTD at room temperature.'''<br />
# Turn off the output of the triple-output power supply.<br />
# Connect the RTD to the electronic breadboard.<br />
# '''Measure the resistance of the 15 k&Omega; resistor with a DMM and record the value in your lab report.'''<br />
# Connect the 15k&Omega; resistor in series with the RTD.<br />
# Set the power supply for '''SERIES''' operation.<br />
# Set the output voltage to 15 V on the supply and make note of its exact value using the DMM.<br />
# Set the current limit to 0.1 A.<br />
# Connect the positive terminal of '''CH1''' to a banana terminal on the breadboard for +15 V and wire it to the top red bus strip for +15 V supply.<br />
# Connect the negative terminal of '''CH2''' to a banana terminal on the breadboard for -15 V.<br />
# Connect the positive terminal of '''CH2''' to the black terminal on the breadboard for a ground reference and wire it to the top blue bus strip.<br />
#* Use three jump wires to minimize the parasitic voltage drop due to the breadboard tie points.<br />
# Connect the +15 V bus strip to the second vertical, red bus strip and wire this bus strip to the top of your 15 k&Omega; resistor.<br />
# Turn on the output of the power supply and measure the voltage across the RTD using the DMM.<br />
# '''What is the measured voltage of the RTD at room temperature and how does it compare to your predictions above?'''<br />
# '''What is the temperature, based on your measured voltage and how does it compare with the clock on the wall (or some better measure)?'''<br />
# '''How is the temperature calculation affected by a 1% change in the 15 V input voltage?'''<br />
<br />
====Photodiode amplifier circuit====<br />
<br />
The current produced by the photodiode detector is a few tens of nanoamperes. This very small current must be amplified to a level where it can be easily measured. The data acquisition system you will use can measure voltages between + and -10 V. Use the two-stage op amp circuit shown below to convert the photodiode current to a voltage.<br />
<br />
<center>[[Image:Part1_TransimpedanceAmplifier.jpg|700 px|thumb|center|Multi-stage photodiode amplification circuit]]</center><br />
<br />
You will have to decide how to distribute the gain between the two op amp stages in the circuit. It is best to put as much gain in early stages of a multi-stage amplifier as possible. However, when the gain of a single stage becomes very high, nonidealities of the op amp become bothersome. The first stage of the amplifier will behave reasonably well as long as the gain is less than about 10<sup>6</sup> &Omega;.<br />
<br />
# '''Analyze the photodiode amplifier circuit. Choose resistor values to achieve an overall gain that will yield an output of about 5 V when the input is 500 nA.'''<br />
# Construct the circuit on your breadboard.<br />
#* Disable the power supply before you build.<br />
#* Do not yet connect the photodiode.<br />
#* Orient the LF411 op-amps so that pin 1, which is marked with a dot on the package, is at the upper left.<br />
# Connect the +/- 15 V bus strips to power the op-amps power pins.<br />
# Connect the vertical blue bus strips to the horizontal blue bus strip.<br />
<br />
=====Test connectivity and basic operation=====<br />
<br />
# Verify that all your component connections are correct and match the design.<br />
# Check that wires and components are properly seated in the breadboard and not suspiciously loose.<br />
#* You can use the ohm-meter function of the digital multi-meter to verify continuity.<br />
# Turn on the power supply and verify that +15V appears on pin 7 and -15V on pin 4.<br />
# '''Short pins 2 and 3 (the V- and V+ inputs to the op-amp) with a small wire and measure the output voltages of IC1 and IC2.'''<br />
#* With the photodiode shorted the current signal is zero, but you may still see a significant offset voltage from 50 to 100 mV at IC1 pin 6.<br />
#* Refer to [http://measure.mit.edu/~20.309/wiki/index.php?title=Real_electronics#Real_op-amps Real Electronics, real op-amps] or ask an instructor to understand and address this non-ideal behavior.<br />
<br />
==Data acquisition hardware subsystem==<br />
<br />
{{:20.309:DAQ System}}<br />
<br />
====Summary of DAQ inputs/outputs====<br />
<br />
A special connector has been prepared for you, connected as shown below. The connections and cable wire colors are also summarized [http://dl.dropbox.com/u/12957607/DAQ_Cable_Assembly_for_DNA_melting_lab.pdf in a memo].<br />
<br />
{| class="wikitable"<br />
|-<br />
! Signal Name<br />
! Signal Location<br />
! Ground Location<br />
! Pin wire color<br />
|-<br />
! DAQ Inputs <br />
|-<br />
! RTD<br />
| AI0+<br />
| AI0-<br />
| +Orange / -Black [lone pair, not with third (red) wire]<br />
|-<br />
! Photodiode <br />
| AI1+ <br />
| AI1-<br />
| +Green / -White<br />
|}<br />
<br />
<center>[[Image:DAQ_Wire_Colors.png|450 px|thumb|center|DAQ Connection Cable]]</center><br />
<br />
==DNAMelter software==<br />
<br />
[[Image:BasicDNAMelterIcon.png|75 px|left]]<br />
Use the <code>Basic DNA Melter GUI</code> program (located on the lab computer desktop) to collect data from the apparatus.<br />
<br />
At the end of an experimental run, use the "Save" button to write the date to a file. The data will be tab-delimited and can be read into Matlab with the <code>load</code> command.<br />
<br />
Documentation for the DNAMelter software is available here: [[DNA Melting: Using the Basic DNAMelter GUI]]<br />
<br />
====If you need to debug the DAQ (skip otherwise!)====<br />
* When debugging problems, it is frequently useful to ascertain whether the data acquisition system is working properly. National Instruments provides an application for this purpose called Measurement and Automation Explorer. Use Measurement and Automation Explorer to verify that the DAQ is properly connected to and communicating with the computer. The program also provides the ability to manually control all of the DAQ system's inputs and outputs.<br />
** Launch Measurement and Automation Explorer by selecting Start->All Applications->National Instruments->Measurement and Automation.<br />
** In the "Configuration" tab, select "Devices and Interfaces" and then "NI-DAQMx Devices." Select "Dev1."<br />
** If Dev 1 does not show up, the problem is that computer cannot communicate with the DAQ system. Ensure that the USB cable is connected to the DAQ system. If the cable is connected and the device still does not show up, unplug the USB cable, count to five, and plug it back in. <br />
** If the DAQ shows up under a different name, disconnect the USB cable. Delete all the "NI USB-6xxx" entries and then reconnect the USB cable. Verify that one entry reappears and that it is called "Dev1." <br />
** Select the "Test Panels" tab to manually control or read signals from the DAQ.<br />
* If you run DNAMelter and there is a data acquisition error, for example, "<code>Data acquisition cannot start!</code>", open Measurement and Automation Explorer and follow the steps above. Run DNAMelter again. If it still doesn't work, grab a beer or ask an instructor for help.<br />
* The DAQ has only one analog-to-digital converter (ADC). A multiplexer circuit sequentially samples voltages from the analog inputs configured in the control software. If any of the inputs is outside the allowable range of +/-10V, all of the inputs may behave erratically. The voltages reported may look reasonable so this can be very misleading. If none of the DAQ readings make sense, ensure that all of the analog voltages are in the allowable range and reset the DAQ by cycling the power of the DAQ (use the switch on the 6341 or disconnect the USB of the 6212).<br />
* If the DAQ gets short-circuited, it may crash. If this happens, Matlab will frequently give a deceptive error message, for example claiming that the limits on the band-pass filter do not satisfy some condition. In this case, correct the connection and cycle the DAQ power.<br />
<br />
==Part 1 measurements==<br />
<br />
Please read this entire section before beginning. Then start at Testing the instrument, with reference back to the sample sections as-needed.<br />
<br />
===Making samples===<br />
<br />
{{Template:Biohazard warning|message=LC Green in DMSO is readily absorbed through skin. Synthetic oligonucleotides may be harmful by inhalation, ingestion, or skin absorption. Wear gloves when handling samples. Wear safety goggles at all times when pipetting the LC Green/DNA samples. Do not create aerosols. The health effects of LC Green have not been thoroughly investigated. See the LC Green and synthetic oligonucleotide under <code>../EHS Guidelines/MSDS Repository</code> in the course locker for more information.}}<br />
<br />
====Sample prep steps====<br />
<br />
#Pipet 500 &mu;L of DNA plus dye solution into a glass vial. <br />
#Pipet up to 200 &mu;L of mineral oil on top of the sample to help prevent evaporation. It will help to pipette into the corners first. Be careful not to get oil on the cuvette sides far above the sample. It will run down during your experiment and cause shifts in the photodiode signal. Place the cap loosely on the vial. <br />
#*Keep the sample vertical to make sure the oil stays on top. <br />
<br />
* You can use the same sample for several heating/cooling cycles.<br />
* Only discard a sample if you lose significant volume due to evaporation or if your signal gets too low. <br />
<br />
To clean your glass vial between samples, flush with alcohol in the waste container and rinse with water at the drain. Suck out residual liquid with the vacuum and drawn Pasteur pipette to the left of the sink.<br />
<br />
====Sample disposal====<br />
<br />
{{Template:Environmental Warning|message=Discard pipette tips with DNA sample residue in the pipette tips or the ''Biohazard Sharps'' container. Do not pour synthetic oligonucleotides with LC Green down the drain. Pour your used samples into the waste container provided in the middle of the wet bench.}}<br />
<br />
===Testing the instrument===<br />
<br />
Once your apparatus is built, use samples of fluorescein (~ 30&mu;M concentration) as a stand-in for LC Green + DNA to test the instrument. Fluorescein is cheap, non-toxic and will not bleach as quickly as LC Green.<br />
<br />
====Making a simple measurement of your instrument's low frequency SNR====<br />
<br />
Carry out a simple estimate of the signal to noise ratio (SNR) of the instrument by comparing the difference between the average signal with a sample present and with water as a null reference. This process requires a vial containing 500 uL of 30 uM fluorescein and a vial containing the same amount of water. <br />
<br />
# Run the <code>Basic DNA Melter GUI</code>. <br />
# Place a vial of DI water in your instrument.<br />
# Clear the data and wait 10 seconds.<br />
# Replace the water vial with the DNA of fluorescein vial, record for another 10 seconds, then save the data. <br />
#* Be sure that all other conditions, such as temperature, are stable throughout the test. <br />
# Segment your data into that when the water was present and that when the fluorescein was present. Calculate the average signal in each case. Record the difference between the two averages as your signal. <br />
# Now discard the water portion of your signal and subtract the average fluorescein signal value from the fluorescein signal.<br />
# Measure the standard deviation of this residual and record as record as your estimate of the noise level.<br />
# '''Calculate the SNR as signal divided by noise and record in your lab report.'''<br />
<br />
===Experiment steps===<br />
<br />
Once the instrument is confirmed to be functioning properly, follow these instructions to generate melting curve data for the 20 bp DNA-LC Green solution.<br />
<br />
#Open and run the <code>Basic DNA Melter GUI</code> in Matlab and follow the instructions there.<br />
# Confirm that block temperature is near room temperature when the Diablotek supply is off.<br />
# Apply heat to the block by switching the Diablotek on to increase the block temperature to 95&deg;C, toggle the Diablotek power in order to hold the temperature long enough for the fluorescence curve to reach a minimum, then allow the block to cool down by switching off the Diablotek.<br />
#* Other approaches may be used. One goal would be to heat the block more slowly, possibly by toggling the current while heating.<br />
# Now prepare a sample using the stock of "beater" DNA.<br />
# Place the vial in the instrument, but keep the LED off until ready to measure the melting curve.<br />
# Measure melting curves of the beater DNA sample as-desired to understand instrument operation.<br />
# Prepare a sample using fresh 20 bp 100 mM KCl DNA.<br />
# Measure a melting curve of the fresh sample.<br />
#* Be sure to save a copy in a local directory.<br />
<br />
{{:DNA Melting Report Requirements for Part 1}}<br />
<br />
==Lab manual sections==<br />
<br />
*[[Lab Manual:Measuring DNA Melting Curves]]<br />
*[[DNA Melting: Simulating DNA Melting - Basics]]<br />
*[[DNA Melting Part 1: Measuring Temperature and Fluorescence]]<br />
*[[DNA Melting Report Requirements for Part 1]]<br />
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]<br />
*[[DNA Melting Report Requirements for Part 2]]<br />
<br />
==References==<br />
<references/><br />
<br />
==Subset of datasheets==<br />
(Many more can be found online or on the course share)<br />
#[http://www.ni.com/pdf/manuals/371931f.pdf National Instruments USB-6212 user manual]<br />
#[http://www.ni.com/pdf/manuals/370784d.pdf National Instruments USB-6341 user manual]<br />
#[http://www.national.com/ds/LF/LF411.pdf Op-amp datasheet]<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/DNA_Melting_Part_1:_Measuring_Temperature_and_FluorescenceDNA Melting Part 1: Measuring Temperature and Fluorescence2015-11-08T18:40:24Z<p>Steven Nagle: /* Optical subsystem */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
<br />
[[Image:DNA Melting Block Diagram.jpg|frame|DNA melting apparatus block diagram showing the five functional groups of the instrument.]]<br />
<br />
<br />
''In theory, there is no difference between theory and practice. But, in practice, there is.'' <br />
<br />
''&mdash;[http://en.wikiquote.org/wiki/Jan_L._A._van_de_Snepscheut Jan L. A. van de Snepscheut]/[http://en.wikiquote.org/wiki/Yogi_Berra Yogi Berra]/and or [http://c2.com/cgi/wiki?DifferenceBetweenTheoryAndPractice Chuck Reid]'' <br />
<br />
You will construct a basic DNA melting instrument consisting of the five functional groups shown in the block diagram:<br />
* excitation<br />
* heating<br />
* fluorescent detection<br />
* temperature measurement<br />
* data acquisition and control<br />
<br />
A metal block will hold a sample vial in the excitation light path and transfer heat to the sample. The sample contains a combination of DNA, SYBR Green I and a salt. Recall that SYBR Green I fluoresces when it is bound to double stranded DNA (dsDNA) by a factor of up to 1000x compared to when it is bound to single stranded DNA (ssDNA). Detection of this fluorescence provides a relative measure of the fraction of dsDNA to total DNA, which can be compared to predictions from a thermodynamic model.<br />
<br />
The dye has a peak sensitivity to blue light at 497 nm so a blue LED will be used to illuminate the sample. The dye emits green light with an emission peak at 520 nm. A photodiode will be placed at 90&deg; to the LED source to detect the green light emitted by SYBR Green I. Because LEDs have a fairly broad spectrum, first a moderately narrow band-pass filter will be used to limit the excitation wavelength to between 450 and 490 nm. Then a complimentary long-pass emission filter will be placed between the sample and detector to pass only wavelengths longer than 515 nm. This filter combination, when used with this dye system, eliminates any background signal due to the excitation light.<br />
<br />
The sample block will be heated by a TEC (thermoelectric cooler) and its temperature measured by an embedded RTD (resistance temperature detector). Also, since photodiodes produce a very small amount of current, a high gain amplifier will convert the photodiode current into a measurable voltage. The DAQ (data acquisition card) and PC will digitize the amplified photodiode and RTD signals. A Matlab program is provided to record these fluorescence and temperature signals over time and save the data to a file. The file can then be loaded into Matlab for analysis, or if you prefer, Python or equivalent where the signals will be converted into dsDNA concentration and sample temperature. Finally, the dsDNA sample melting temperature will be estimated from a plot of df/dT vs T (''f'' represents the fraction of total DNA that is in double-stranded form) and from a comparison to the thermodynamic model.<br />
<br />
The functional groups in the block diagram are one of two fundamental frameworks used to describe an instrument. The other sub-divides the instrument into categories that often mirror historical areas of engineering. The former is focused on what will be done, including the overall scientific goals and the physical processes to be measured. The latter is focused on how it will be done, e.g. using optics, electronics or perhaps a practical knowledge of thermal management. A good engineer will constantly be thinking of the instrument in both frameworks. <br />
<br />
==Mechanical and thermal components==<br />
<br />
[[Image:DNA_Setup_v2_Iso.png|600 px|thumb|center|DNA Melting System Model]]<br />
<br />
Mechanical support for the heating block, sample and optics will be built on a portable optical breadboard from [http://www.thorlabs.com ThorLabs]. Most of the components in the lab are from this supplier because the selection is large, the quality is quite good and the price reasonable. Although you are free to modify the design as the lab progresses, begin a build from the standard design shown above and demonstrated by the example in the lab. Gather the following Thorlabs components but do not start to assemble.<br />
<br />
* 1 MB1224 optical breadboard<br />
* 2 CL5 clamps (or similar)<br />
* 2 1/4-20 5/8" screws<br />
* 4 1/4-20 1/4" screws<br />
<br />
The thermal subsystem includes all components to control heat flow into and out of the sample and monitor its temperature. Heating is accomplished using thermal electric coolers (TECs) in reverse. The hot side is placed in direct contact with the heating block, which is in direct contact with the sample vial. The TECs will be powered by manually controlling the Diablotek power supply. To avoid contamination of the sample or perturbation of its temperature, the temperature of a resistive temperature detector (RTD) embedded in the heating block will be monitored as a proxy for the sample temperature. In this part of the lab, air convection will cool down the sample when the Diablotek is switched off. The heat sink will help and the fan may be connected to a triple-output power supply to help, but these are included primarily for use in Part 2.<br />
<br />
===Parts===<br />
Gather the following components from the bins on the Center Cabinets, the DNA Melting drawer at the wet bench or on the East Cabinets:<br />
<br />
[[Image:CenterCabinets.jpg|right|250px|thumb|'''Center Cabinets''' The bins for most parts that you need will be set out on top of these cabinets, roughly in the center of the lab.]]<br />
[[Image:WetBench.jpg|right|250px|thumb|'''Wet Bench''' Supplies for our wet bench can be found in the three drawers at the left of the bench, the drawer left of the sink and on the wall behind and to the left as you face the wet bench. The drawer at the right of the wet bench contains various supplies for the DNA Melting instrument.]]<br />
[[Image:EastCabinets.jpg|right|250px|thumb|'''East Cabinets''' The center shelves of the East Cabinets contain the electronic breadboards, breadboard jumper wire, TEC connectors and TEC power supplies. Often these supplies are laid out on top of these cabinets.]]<br />
<br />
* These components are already assembled for you: A metal support bracket, having a fan, heatsink, 2 TEC heaters/coolers, and a heating block attached to it<br />
* 3 wire nuts<br />
* 1 Diablotek 250 W power supply (at your station or on a shelf above your station)<br />
* 1 red/black TEC extension wire assembly (in a box on the ''''Center Cabinets'''')<br />
<br />
===Assembly instructions===<br />
<br />
[[Image:TEC_picture.jpg|150 px|thumb|right|TEC showing hot side up when current flows into the red lead]]<br />
<br />
Ensure that the following have been done/attached to your metal bracket:<br />
# The bracket, TECs, fan and heatsink should already be assembled for you.<br />
# Verify that the colors of the leads match. If not, flip one TEC.<br />
# Connect the black lead of the top TEC to the red lead of the bottom TEC using a wire nut.<br />
# Connect the TEC to the extension wire assembly using a wire nut to join the white wire to the red lead of the top TEC and the black wire to the black lead on the bottom TEC.<br />
# Join the white connector on the assembly to the black 2x2 connector of the Diabloteck.<br />
# Test the TECs by holding the them between thumb and finger while briefly flipping the switch on the Diablotek.<br />
#* One side should immediately heat up while the other immediately cools.<br />
#* The top side should be the hot side when the red wires extend down and to the '''left'''.<br />
#* If the Diablotek immediately shuts off (the fan will stop spinning) then there is a short. Check all connections.<br />
# Once the TEC assembly checks out, butter the bottom side of the assembly and place that side on the heat sink with the leads facing to the right.<br />
# Finally, apply thermal grease to the top side of the TEC assembly, place the heating block on top and anchor to the heat sink using the 1/2" long 4-40 screws and plastic washers.<br />
<br />
==Optical subsystem==<br />
<br />
A good optical subsystem design will be compact, stable and simple. It includes the optical components of the excitation and fluorescence detection functional groups from the block diagram. A good design will efficiently illuminate the sample and collect the maximum possible amount of emitted fluorescent light. There are a variety of ways to construct the apparatus. Use the model above and the example system in the lab as guides.<br />
<br />
The following is a set of instructions if you're not clear on how to replicate the example system in the lab. In addition to the following parts, you might want to add a few lenses to collimate your LED onto your sample, and to focus the light coming from your sample onto your photodiode. <br />
<br />
* 2 PH2 post holders<br />
* 2 BA1 post holder bases<br />
* 2 1/4-20 1/4" screws<br />
* 2 1/4-20 5/8" screws<br />
* 2 1/4" washers<br />
* 4 CP02 cage plates<br />
* 2 8-32 1/2" setscrews<br />
* 2 TR2 posts<br />
* 6 ER3 cage rails<br />
* 12 4-40 1/4" setscrews (or similar length)<br />
* 4 SML05 lens tubes<br />
* 4 SM1LTRR retaining rings<br />
* 1 blue LED PCB<br />
* 1 D470/40 excitation filter<br />
* 1 E515LPv2 emission filter<br />
* 1 SM1A6 adapter between SM1 and SM05 threads<br />
* 1 SM05PD1A mounted photodiode<br />
<br />
===Assembly instructions===<br />
Use an Instructor instrument or the Solid Works model above as guide to assemble your optical subassembly. Note also that these instructions are also a guid. If you wish to build your own personal design, please do.<br />
<br />
# Attach each PH2 to a BA1 using 1/4" screws.<br />
# Attach two of the CP02 cage plates to two TR2 posts using an 8-32 setscrew.<br />
# Insert the TR2/CP02 assembly into the PH2/BA1 assemblies and lock in place with the TS25H setscrews.<br />
# Insert three ER4 cage rails into each CP02 above and lock in place with SS4S025 set screws or similar.<br />
#* Leave the blank hole on the bottom of the assembly.<br />
# Attach one of the remaining CP02 cage plates on the free end of each assembly above and lock in place.<br />
# Attach each of the above assemblies to the MB1224 using a washer and 1/4-20 screw less than 1" in length.<br />
# Arrange on the MB1224 and adjust CP02 heights as desired.<br />
# Mount the LED PCB in an SML05 lens tube and fix with a retaining ring.<br />
# Mount the excitation and emission filters in each of two lens tubes and fix with retaining ring.<br />
#* The arrow label on the thickness of the filter points ''toward the light source''.<br />
# Thread the SM1A6 adapter into a lens tube and fix with a retaining ring.<br />
# Thread the photodiode into the SM1A6 adapter.<br />
#* Teflon pipe thread tape can be used to hold the photodiode in the desired position.<br />
# Mount each lens tube in the appropriate CP02 cage plates.<br />
# Arrange each the resultant assemblies on the breadboard to most fully illuminate the sample vial and to gather the most of the resultant light on the photodiode.<br />
<br />
==Electronics subsystem==<br />
<br />
The electrical subsystem includes the LED with a current-limiting resistor, the photodiode, the transimpedance amplifier and a temperature detection circuit. The remainder of this section provides an example of each of these systems and instructions to build and test each one. Nevertheless, alternate ideas are welcome. Feel free to discuss with the Instructors.<br />
<br />
===Parts===<br />
<br />
Gather the following components from the electronics drawer to the right of the optics drawers, from the counter top opposite the wet bench and from the cable organizer on the East wall to the right of the white board.<br />
* 1 electronic breadboard<br />
* 1 jump wire kit<br />
* 2 red 3' banana cable or similar<br />
* 1 black 3' banana cable or similar<br />
* individual wire, as-needed from the scrap wire box or the wire spools opposite the wet bench<br />
* 1 62 &Omega; resistor from the center counter top on the South wall<br />
* 1 15 k&Omega; resistor<br />
* other resistors, as-needed for the amplifier<br />
* 1 blue LED from the DNA Melting drawer at the far right of the wet bench<br />
* 2 LF411 op-amps from the electronics drawer<br />
* 4 capacitors between 0.1 and 22 uF from either the electronics drawer or the counter top where the screws are stored.<br />
<br />
===Assembly instructions===<br />
<br />
====LED circuit====<br />
The led circuit consists of a 62 &Omega; resistor in series with a blue, jumbo LED. The circuit will be powered by the 5 volt fixed output of the lab power supply. The 62 &Omega; resistor prevents excessive current from flowing through the LED. Excessive current will let the smoke out of the LED.<br />
<br />
# Build the LED circuit on your electronic breadboard.<br />
#* Don't forget to connect the resistor. Directly connecting the LED to 5 V will blow out the LED.<br />
# Disable the output of the lab power supply.<br />
# Connect the LED circuit to the fixed 5 V output.<br />
<br />
====Temperature detection circuit====<br />
A platinum RTD is embedded inside the heating block. The resistance of the RTD varies with temperature according to the equation ''R<sub>RTD</sub>''=1000 &Omega; + 3.85 ''&theta;'', where ''&theta;'' is the temperature of the RTD in degrees Celsius. The easiest way to make a temperature measurement is to connect the RTD in series with a fixed resistor and a power supply. The output voltage of the resulting divider circuit varies as a function of temperature. <br />
<br />
The maximum current of the RTD is 1 mA. You will use a 15 V supply to the circuit, so the resistor must be at least 14 k&Omega;. Use a 15 k&Omega; resistor.<br />
<br />
# '''Find an equation for ''V<sub>out</sub>'' as a function of temperature. What voltage do you expect given an input voltage of 15 V with the RTD at room temperature.'''<br />
# Turn off the output of the triple-output power supply.<br />
# Connect the RTD to the electronic breadboard.<br />
# '''Measure the resistance of the 15 k&Omega; resistor with a DMM and record the value in your lab report.'''<br />
# Connect the 15k&Omega; resistor in series with the RTD.<br />
# Set the power supply for '''SERIES''' operation.<br />
# Set the output voltage to 15 V on the supply and make note of its exact value using the DMM.<br />
# Set the current limit to 0.1 A.<br />
# Connect the positive terminal of '''CH1''' to a banana terminal on the breadboard for +15 V and wire it to the top red bus strip for +15 V supply.<br />
# Connect the negative terminal of '''CH2''' to a banana terminal on the breadboard for -15 V.<br />
# Connect the positive terminal of '''CH2''' to the black terminal on the breadboard for a ground reference and wire it to the top blue bus strip.<br />
#* Use three jump wires to minimize the parasitic voltage drop due to the breadboard tie points.<br />
# Connect the +15 V bus strip to the second vertical, red bus strip and wire this bus strip to the top of your 15 k&Omega; resistor.<br />
# Turn on the output of the power supply and measure the voltage across the RTD using the DMM.<br />
# '''What is the measured voltage of the RTD at room temperature and how does it compare to your predictions above?'''<br />
# '''What is the temperature, based on your measured voltage and how does it compare with the clock on the wall (or some better measure)?'''<br />
# '''How is the temperature calculation affected by a 1% change in the 15 V input voltage?'''<br />
<br />
====Photodiode amplifier circuit====<br />
<br />
The current produced by the photodiode detector is a few tens of nanoamperes. This very small current must be amplified to a level where it can be easily measured. The data acquisition system you will use can measure voltages between + and -10 V. Use the two-stage op amp circuit shown below to convert the photodiode current to a voltage.<br />
<br />
<center>[[Image:Part1_TransimpedanceAmplifier.jpg|700 px|thumb|center|Multi-stage photodiode amplification circuit]]</center><br />
<br />
You will have to decide how to distribute the gain between the two op amp stages in the circuit. It is best to put as much gain in early stages of a multi-stage amplifier as possible. However, when the gain of a single stage becomes very high, nonidealities of the op amp become bothersome. The first stage of the amplifier will behave reasonably well as long as the gain is less than about 10<sup>6</sup> &Omega;.<br />
<br />
# '''Analyze the photodiode amplifier circuit. Choose resistor values to achieve an overall gain that will yield an output of about 5 V when the input is 500 nA.'''<br />
# Construct the circuit on your breadboard.<br />
#* Disable the power supply before you build.<br />
#* Do not yet connect the photodiode.<br />
#* Orient the LF411 op-amps so that pin 1, which is marked with a dot on the package, is at the upper left.<br />
# Connect the +/- 15 V bus strips to power the op-amps power pins.<br />
# Connect the vertical blue bus strips to the horizontal blue bus strip.<br />
<br />
=====Test connectivity and basic operation=====<br />
<br />
# Verify that all your component connections are correct and match the design.<br />
# Check that wires and components are properly seated in the breadboard and not suspiciously loose.<br />
#* You can use the ohm-meter function of the digital multi-meter to verify continuity.<br />
# Turn on the power supply and verify that +15V appears on pin 7 and -15V on pin 4.<br />
# '''Short pins 2 and 3 (the V- and V+ inputs to the op-amp) with a small wire and measure the output voltages of IC1 and IC2.'''<br />
#* With the photodiode shorted the current signal is zero, but you may still see a significant offset voltage from 50 to 100 mV at IC1 pin 6.<br />
#* Refer to [http://measure.mit.edu/~20.309/wiki/index.php?title=Real_electronics#Real_op-amps Real Electronics, real op-amps] or ask an instructor to understand and address this non-ideal behavior.<br />
<br />
==Data acquisition hardware subsystem==<br />
<br />
{{:20.309:DAQ System}}<br />
<br />
====Summary of DAQ inputs/outputs====<br />
<br />
A special connector has been prepared for you, connected as shown below. The connections and cable wire colors are also summarized [http://dl.dropbox.com/u/12957607/DAQ_Cable_Assembly_for_DNA_melting_lab.pdf in a memo].<br />
<br />
{| class="wikitable"<br />
|-<br />
! Signal Name<br />
! Signal Location<br />
! Ground Location<br />
! Pin wire color<br />
|-<br />
! DAQ Inputs <br />
|-<br />
! RTD<br />
| AI0+<br />
| AI0-<br />
| +Orange / -Black [lone pair, not with third (red) wire]<br />
|-<br />
! Photodiode <br />
| AI1+ <br />
| AI1-<br />
| +Green / -White<br />
|}<br />
<br />
<center>[[Image:DAQ_Wire_Colors.png|450 px|thumb|center|DAQ Connection Cable]]</center><br />
<br />
==DNAMelter software==<br />
<br />
[[Image:BasicDNAMelterIcon.png|75 px|left]]<br />
Use the <code>Basic DNA Melter GUI</code> program (located on the lab computer desktop) to collect data from the apparatus.<br />
<br />
At the end of an experimental run, use the "Save" button to write the date to a file. The data will be tab-delimited and can be read into Matlab with the <code>load</code> command.<br />
<br />
Documentation for the DNAMelter software is available here: [[DNA Melting: Using the Basic DNAMelter GUI]]<br />
<br />
====If you need to debug the DAQ (skip otherwise!)====<br />
* When debugging problems, it is frequently useful to ascertain whether the data acquisition system is working properly. National Instruments provides an application for this purpose called Measurement and Automation Explorer. Use Measurement and Automation Explorer to verify that the DAQ is properly connected to and communicating with the computer. The program also provides the ability to manually control all of the DAQ system's inputs and outputs.<br />
** Launch Measurement and Automation Explorer by selecting Start->All Applications->National Instruments->Measurement and Automation.<br />
** In the "Configuration" tab, select "Devices and Interfaces" and then "NI-DAQMx Devices." Select "Dev1."<br />
** If Dev 1 does not show up, the problem is that computer cannot communicate with the DAQ system. Ensure that the USB cable is connected to the DAQ system. If the cable is connected and the device still does not show up, unplug the USB cable, count to five, and plug it back in. <br />
** If the DAQ shows up under a different name, disconnect the USB cable. Delete all the "NI USB-6xxx" entries and then reconnect the USB cable. Verify that one entry reappears and that it is called "Dev1." <br />
** Select the "Test Panels" tab to manually control or read signals from the DAQ.<br />
* If you run DNAMelter and there is a data acquisition error, for example, "<code>Data acquisition cannot start!</code>", open Measurement and Automation Explorer and follow the steps above. Run DNAMelter again. If it still doesn't work, grab a beer or ask an instructor for help.<br />
* The DAQ has only one analog-to-digital converter (ADC). A multiplexer circuit sequentially samples voltages from the analog inputs configured in the control software. If any of the inputs is outside the allowable range of +/-10V, all of the inputs may behave erratically. The voltages reported may look reasonable so this can be very misleading. If none of the DAQ readings make sense, ensure that all of the analog voltages are in the allowable range and reset the DAQ by cycling the power of the DAQ (use the switch on the 6341 or disconnect the USB of the 6212).<br />
* If the DAQ gets short-circuited, it may crash. If this happens, Matlab will frequently give a deceptive error message, for example claiming that the limits on the band-pass filter do not satisfy some condition. In this case, correct the connection and cycle the DAQ power.<br />
<br />
==Part 1 measurements==<br />
<br />
Please read this entire section before beginning. Then start at Testing the instrument, with reference back to the sample sections as-needed.<br />
<br />
===Making samples===<br />
<br />
{{Template:Biohazard warning|message=LC Green in DMSO is readily absorbed through skin. Synthetic oligonucleotides may be harmful by inhalation, ingestion, or skin absorption. Wear gloves when handling samples. Wear safety goggles at all times when pipetting the LC Green/DNA samples. Do not create aerosols. The health effects of LC Green have not been thoroughly investigated. See the LC Green and synthetic oligonucleotide under <code>../EHS Guidelines/MSDS Repository</code> in the course locker for more information.}}<br />
<br />
====Sample prep steps====<br />
<br />
#Pipet 500 &mu;L of DNA plus dye solution into a glass vial. <br />
#Pipet up to 200 &mu;L of mineral oil on top of the sample to help prevent evaporation. It will help to pipette into the corners first. Be careful not to get oil on the cuvette sides far above the sample. It will run down during your experiment and cause shifts in the photodiode signal. Place the cap loosely on the vial. <br />
#*Keep the sample vertical to make sure the oil stays on top. <br />
<br />
* You can use the same sample for several heating/cooling cycles.<br />
* Only discard a sample if you lose significant volume due to evaporation or if your signal gets too low. <br />
<br />
To clean your glass vial between samples, flush with alcohol in the waste container and rinse with water at the drain. Suck out residual liquid with the vacuum and drawn Pasteur pipette to the left of the sink.<br />
<br />
====Sample disposal====<br />
<br />
{{Template:Environmental Warning|message=Discard pipette tips with DNA sample residue in the pipette tips or the ''Biohazard Sharps'' container. Do not pour synthetic oligonucleotides with LC Green down the drain. Pour your used samples into the waste container provided in the middle of the wet bench.}}<br />
<br />
===Testing the instrument===<br />
<br />
Once your apparatus is built, use samples of fluorescein (~ 30&mu;M concentration) as a stand-in for LC Green + DNA to test the instrument. Fluorescein is cheap, non-toxic and will not bleach as quickly as LC Green.<br />
<br />
====Making a simple measurement of your instrument's low frequency SNR====<br />
<br />
Carry out a simple estimate of the signal to noise ratio (SNR) of the instrument by comparing the difference between the average signal with a sample present and with water as a null reference. This process requires a vial containing 500 uL of 30 uM fluorescein and a vial containing the same amount of water. <br />
<br />
# Run the <code>Basic DNA Melter GUI</code>. <br />
# Place a vial of DI water in your instrument.<br />
# Clear the data and wait 10 seconds.<br />
# Replace the water vial with the DNA of fluorescein vial, record for another 10 seconds, then save the data. <br />
#* Be sure that all other conditions, such as temperature, are stable throughout the test. <br />
# Segment your data into that when the water was present and that when the fluorescein was present. Calculate the average signal in each case. Record the difference between the two averages as your signal. <br />
# Now discard the water portion of your signal and subtract the average fluorescein signal value from the fluorescein signal.<br />
# Measure the standard deviation of this residual and record as record as your estimate of the noise level.<br />
# '''Calculate the SNR as signal divided by noise and record in your lab report.'''<br />
<br />
===Experiment steps===<br />
<br />
Once the instrument is confirmed to be functioning properly, follow these instructions to generate melting curve data for the 20 bp DNA-LC Green solution.<br />
<br />
#Open and run the <code>Basic DNA Melter GUI</code> in Matlab and follow the instructions there.<br />
# Confirm that block temperature is near room temperature when the Diablotek supply is off.<br />
# Apply heat to the block by switching the Diablotek on to increase the block temperature to 95&deg;C, toggle the Diablotek power in order to hold the temperature long enough for the fluorescence curve to reach a minimum, then allow the block to cool down by switching off the Diablotek.<br />
#* Other approaches may be used. One goal would be to heat the block more slowly, possibly by toggling the current while heating.<br />
# Now prepare a sample using the stock of "beater" DNA.<br />
# Place the vial in the instrument, but keep the LED off until ready to measure the melting curve.<br />
# Measure melting curves of the beater DNA sample as-desired to understand instrument operation.<br />
# Prepare a sample using fresh 20 bp 100 mM KCl DNA.<br />
# Measure a melting curve of the fresh sample.<br />
#* Be sure to save a copy in a local directory.<br />
<br />
{{:DNA Melting Report Requirements for Part 1}}<br />
<br />
==Lab manual sections==<br />
<br />
*[[Lab Manual:Measuring DNA Melting Curves]]<br />
*[[DNA Melting: Simulating DNA Melting - Basics]]<br />
*[[DNA Melting Part 1: Measuring Temperature and Fluorescence]]<br />
*[[DNA Melting Report Requirements for Part 1]]<br />
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]<br />
*[[DNA Melting Report Requirements for Part 2]]<br />
<br />
==References==<br />
<references/><br />
<br />
==Subset of datasheets==<br />
(Many more can be found online or on the course share)<br />
#[http://www.ni.com/pdf/manuals/371931f.pdf National Instruments USB-6212 user manual]<br />
#[http://www.ni.com/pdf/manuals/370784d.pdf National Instruments USB-6341 user manual]<br />
#[http://www.national.com/ds/LF/LF411.pdf Op-amp datasheet]<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/DNA_Melting_Part_1:_Measuring_Temperature_and_FluorescenceDNA Melting Part 1: Measuring Temperature and Fluorescence2015-11-08T18:39:55Z<p>Steven Nagle: /* Assembly instructions */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
<br />
[[Image:DNA Melting Block Diagram.jpg|frame|DNA melting apparatus block diagram showing the five functional groups of the instrument.]]<br />
<br />
<br />
''In theory, there is no difference between theory and practice. But, in practice, there is.'' <br />
<br />
''&mdash;[http://en.wikiquote.org/wiki/Jan_L._A._van_de_Snepscheut Jan L. A. van de Snepscheut]/[http://en.wikiquote.org/wiki/Yogi_Berra Yogi Berra]/and or [http://c2.com/cgi/wiki?DifferenceBetweenTheoryAndPractice Chuck Reid]'' <br />
<br />
You will construct a basic DNA melting instrument consisting of the five functional groups shown in the block diagram:<br />
* excitation<br />
* heating<br />
* fluorescent detection<br />
* temperature measurement<br />
* data acquisition and control<br />
<br />
A metal block will hold a sample vial in the excitation light path and transfer heat to the sample. The sample contains a combination of DNA, SYBR Green I and a salt. Recall that SYBR Green I fluoresces when it is bound to double stranded DNA (dsDNA) by a factor of up to 1000x compared to when it is bound to single stranded DNA (ssDNA). Detection of this fluorescence provides a relative measure of the fraction of dsDNA to total DNA, which can be compared to predictions from a thermodynamic model.<br />
<br />
The dye has a peak sensitivity to blue light at 497 nm so a blue LED will be used to illuminate the sample. The dye emits green light with an emission peak at 520 nm. A photodiode will be placed at 90&deg; to the LED source to detect the green light emitted by SYBR Green I. Because LEDs have a fairly broad spectrum, first a moderately narrow band-pass filter will be used to limit the excitation wavelength to between 450 and 490 nm. Then a complimentary long-pass emission filter will be placed between the sample and detector to pass only wavelengths longer than 515 nm. This filter combination, when used with this dye system, eliminates any background signal due to the excitation light.<br />
<br />
The sample block will be heated by a TEC (thermoelectric cooler) and its temperature measured by an embedded RTD (resistance temperature detector). Also, since photodiodes produce a very small amount of current, a high gain amplifier will convert the photodiode current into a measurable voltage. The DAQ (data acquisition card) and PC will digitize the amplified photodiode and RTD signals. A Matlab program is provided to record these fluorescence and temperature signals over time and save the data to a file. The file can then be loaded into Matlab for analysis, or if you prefer, Python or equivalent where the signals will be converted into dsDNA concentration and sample temperature. Finally, the dsDNA sample melting temperature will be estimated from a plot of df/dT vs T (''f'' represents the fraction of total DNA that is in double-stranded form) and from a comparison to the thermodynamic model.<br />
<br />
The functional groups in the block diagram are one of two fundamental frameworks used to describe an instrument. The other sub-divides the instrument into categories that often mirror historical areas of engineering. The former is focused on what will be done, including the overall scientific goals and the physical processes to be measured. The latter is focused on how it will be done, e.g. using optics, electronics or perhaps a practical knowledge of thermal management. A good engineer will constantly be thinking of the instrument in both frameworks. <br />
<br />
==Mechanical and thermal components==<br />
<br />
[[Image:DNA_Setup_v2_Iso.png|600 px|thumb|center|DNA Melting System Model]]<br />
<br />
Mechanical support for the heating block, sample and optics will be built on a portable optical breadboard from [http://www.thorlabs.com ThorLabs]. Most of the components in the lab are from this supplier because the selection is large, the quality is quite good and the price reasonable. Although you are free to modify the design as the lab progresses, begin a build from the standard design shown above and demonstrated by the example in the lab. Gather the following Thorlabs components but do not start to assemble.<br />
<br />
* 1 MB1224 optical breadboard<br />
* 2 CL5 clamps (or similar)<br />
* 2 1/4-20 5/8" screws<br />
* 4 1/4-20 1/4" screws<br />
<br />
The thermal subsystem includes all components to control heat flow into and out of the sample and monitor its temperature. Heating is accomplished using thermal electric coolers (TECs) in reverse. The hot side is placed in direct contact with the heating block, which is in direct contact with the sample vial. The TECs will be powered by manually controlling the Diablotek power supply. To avoid contamination of the sample or perturbation of its temperature, the temperature of a resistive temperature detector (RTD) embedded in the heating block will be monitored as a proxy for the sample temperature. In this part of the lab, air convection will cool down the sample when the Diablotek is switched off. The heat sink will help and the fan may be connected to a triple-output power supply to help, but these are included primarily for use in Part 2.<br />
<br />
===Parts===<br />
Gather the following components from the bins on the Center Cabinets, the DNA Melting drawer at the wet bench or on the East Cabinets:<br />
<br />
[[Image:CenterCabinets.jpg|right|250px|thumb|'''Center Cabinets''' The bins for most parts that you need will be set out on top of these cabinets, roughly in the center of the lab.]]<br />
[[Image:WetBench.jpg|right|250px|thumb|'''Wet Bench''' Supplies for our wet bench can be found in the three drawers at the left of the bench, the drawer left of the sink and on the wall behind and to the left as you face the wet bench. The drawer at the right of the wet bench contains various supplies for the DNA Melting instrument.]]<br />
[[Image:EastCabinets.jpg|right|250px|thumb|'''East Cabinets''' The center shelves of the East Cabinets contain the electronic breadboards, breadboard jumper wire, TEC connectors and TEC power supplies. Often these supplies are laid out on top of these cabinets.]]<br />
<br />
* These components are already assembled for you: A metal support bracket, having a fan, heatsink, 2 TEC heaters/coolers, and a heating block attached to it<br />
* 3 wire nuts<br />
* 1 Diablotek 250 W power supply (at your station or on a shelf above your station)<br />
* 1 red/black TEC extension wire assembly (in a box on the ''''Center Cabinets'''')<br />
<br />
===Assembly instructions===<br />
<br />
[[Image:TEC_picture.jpg|150 px|thumb|right|TEC showing hot side up when current flows into the red lead]]<br />
<br />
Ensure that the following have been done/attached to your metal bracket:<br />
# The bracket, TECs, fan and heatsink should already be assembled for you.<br />
# Verify that the colors of the leads match. If not, flip one TEC.<br />
# Connect the black lead of the top TEC to the red lead of the bottom TEC using a wire nut.<br />
# Connect the TEC to the extension wire assembly using a wire nut to join the white wire to the red lead of the top TEC and the black wire to the black lead on the bottom TEC.<br />
# Join the white connector on the assembly to the black 2x2 connector of the Diabloteck.<br />
# Test the TECs by holding the them between thumb and finger while briefly flipping the switch on the Diablotek.<br />
#* One side should immediately heat up while the other immediately cools.<br />
#* The top side should be the hot side when the red wires extend down and to the '''left'''.<br />
#* If the Diablotek immediately shuts off (the fan will stop spinning) then there is a short. Check all connections.<br />
# Once the TEC assembly checks out, butter the bottom side of the assembly and place that side on the heat sink with the leads facing to the right.<br />
# Finally, apply thermal grease to the top side of the TEC assembly, place the heating block on top and anchor to the heat sink using the 1/2" long 4-40 screws and plastic washers.<br />
<br />
==Optical subsystem==<br />
<br />
A good optical subsystem design will be compact, stable and simple. It includes the optical components of the excitation and fluorescence detection functional groups from the block diagram. A good design will efficiently illuminate the sample and collect the maximum possible amount of emitted fluorescent light. There are a variety of ways to construct the apparatus. Use the model above and the example system in the lab as guides.<br />
<br />
The following is a rough set of instructions if you're not clear on how to replicate the example system in the lab. In addition to the following parts, you might want to add a few lenses to collimate your LED onto your sample, and to focus the light coming from your sample onto your photodiode. <br />
<br />
* 2 PH2 post holders<br />
* 2 BA1 post holder bases<br />
* 2 1/4-20 1/4" screws<br />
* 2 1/4-20 5/8" screws<br />
* 2 1/4" washers<br />
* 4 CP02 cage plates<br />
* 2 8-32 1/2" setscrews<br />
* 2 TR2 posts<br />
* 6 ER3 cage rails<br />
* 12 4-40 1/4" setscrews (or similar length)<br />
* 4 SML05 lens tubes<br />
* 4 SM1LTRR retaining rings<br />
* 1 blue LED PCB<br />
* 1 D470/40 excitation filter<br />
* 1 E515LPv2 emission filter<br />
* 1 SM1A6 adapter between SM1 and SM05 threads<br />
* 1 SM05PD1A mounted photodiode<br />
<br />
===Assembly instructions===<br />
Use an Instructor instrument or the Solid Works model above as guide to assemble your optical subassembly. Note also that these instructions are also a guid. If you wish to build your own personal design, please do.<br />
<br />
# Attach each PH2 to a BA1 using 1/4" screws.<br />
# Attach two of the CP02 cage plates to two TR2 posts using an 8-32 setscrew.<br />
# Insert the TR2/CP02 assembly into the PH2/BA1 assemblies and lock in place with the TS25H setscrews.<br />
# Insert three ER4 cage rails into each CP02 above and lock in place with SS4S025 set screws or similar.<br />
#* Leave the blank hole on the bottom of the assembly.<br />
# Attach one of the remaining CP02 cage plates on the free end of each assembly above and lock in place.<br />
# Attach each of the above assemblies to the MB1224 using a washer and 1/4-20 screw less than 1" in length.<br />
# Arrange on the MB1224 and adjust CP02 heights as desired.<br />
# Mount the LED PCB in an SML05 lens tube and fix with a retaining ring.<br />
# Mount the excitation and emission filters in each of two lens tubes and fix with retaining ring.<br />
#* The arrow label on the thickness of the filter points ''toward the light source''.<br />
# Thread the SM1A6 adapter into a lens tube and fix with a retaining ring.<br />
# Thread the photodiode into the SM1A6 adapter.<br />
#* Teflon pipe thread tape can be used to hold the photodiode in the desired position.<br />
# Mount each lens tube in the appropriate CP02 cage plates.<br />
# Arrange each the resultant assemblies on the breadboard to most fully illuminate the sample vial and to gather the most of the resultant light on the photodiode.<br />
<br />
==Electronics subsystem==<br />
<br />
The electrical subsystem includes the LED with a current-limiting resistor, the photodiode, the transimpedance amplifier and a temperature detection circuit. The remainder of this section provides an example of each of these systems and instructions to build and test each one. Nevertheless, alternate ideas are welcome. Feel free to discuss with the Instructors.<br />
<br />
===Parts===<br />
<br />
Gather the following components from the electronics drawer to the right of the optics drawers, from the counter top opposite the wet bench and from the cable organizer on the East wall to the right of the white board.<br />
* 1 electronic breadboard<br />
* 1 jump wire kit<br />
* 2 red 3' banana cable or similar<br />
* 1 black 3' banana cable or similar<br />
* individual wire, as-needed from the scrap wire box or the wire spools opposite the wet bench<br />
* 1 62 &Omega; resistor from the center counter top on the South wall<br />
* 1 15 k&Omega; resistor<br />
* other resistors, as-needed for the amplifier<br />
* 1 blue LED from the DNA Melting drawer at the far right of the wet bench<br />
* 2 LF411 op-amps from the electronics drawer<br />
* 4 capacitors between 0.1 and 22 uF from either the electronics drawer or the counter top where the screws are stored.<br />
<br />
===Assembly instructions===<br />
<br />
====LED circuit====<br />
The led circuit consists of a 62 &Omega; resistor in series with a blue, jumbo LED. The circuit will be powered by the 5 volt fixed output of the lab power supply. The 62 &Omega; resistor prevents excessive current from flowing through the LED. Excessive current will let the smoke out of the LED.<br />
<br />
# Build the LED circuit on your electronic breadboard.<br />
#* Don't forget to connect the resistor. Directly connecting the LED to 5 V will blow out the LED.<br />
# Disable the output of the lab power supply.<br />
# Connect the LED circuit to the fixed 5 V output.<br />
<br />
====Temperature detection circuit====<br />
A platinum RTD is embedded inside the heating block. The resistance of the RTD varies with temperature according to the equation ''R<sub>RTD</sub>''=1000 &Omega; + 3.85 ''&theta;'', where ''&theta;'' is the temperature of the RTD in degrees Celsius. The easiest way to make a temperature measurement is to connect the RTD in series with a fixed resistor and a power supply. The output voltage of the resulting divider circuit varies as a function of temperature. <br />
<br />
The maximum current of the RTD is 1 mA. You will use a 15 V supply to the circuit, so the resistor must be at least 14 k&Omega;. Use a 15 k&Omega; resistor.<br />
<br />
# '''Find an equation for ''V<sub>out</sub>'' as a function of temperature. What voltage do you expect given an input voltage of 15 V with the RTD at room temperature.'''<br />
# Turn off the output of the triple-output power supply.<br />
# Connect the RTD to the electronic breadboard.<br />
# '''Measure the resistance of the 15 k&Omega; resistor with a DMM and record the value in your lab report.'''<br />
# Connect the 15k&Omega; resistor in series with the RTD.<br />
# Set the power supply for '''SERIES''' operation.<br />
# Set the output voltage to 15 V on the supply and make note of its exact value using the DMM.<br />
# Set the current limit to 0.1 A.<br />
# Connect the positive terminal of '''CH1''' to a banana terminal on the breadboard for +15 V and wire it to the top red bus strip for +15 V supply.<br />
# Connect the negative terminal of '''CH2''' to a banana terminal on the breadboard for -15 V.<br />
# Connect the positive terminal of '''CH2''' to the black terminal on the breadboard for a ground reference and wire it to the top blue bus strip.<br />
#* Use three jump wires to minimize the parasitic voltage drop due to the breadboard tie points.<br />
# Connect the +15 V bus strip to the second vertical, red bus strip and wire this bus strip to the top of your 15 k&Omega; resistor.<br />
# Turn on the output of the power supply and measure the voltage across the RTD using the DMM.<br />
# '''What is the measured voltage of the RTD at room temperature and how does it compare to your predictions above?'''<br />
# '''What is the temperature, based on your measured voltage and how does it compare with the clock on the wall (or some better measure)?'''<br />
# '''How is the temperature calculation affected by a 1% change in the 15 V input voltage?'''<br />
<br />
====Photodiode amplifier circuit====<br />
<br />
The current produced by the photodiode detector is a few tens of nanoamperes. This very small current must be amplified to a level where it can be easily measured. The data acquisition system you will use can measure voltages between + and -10 V. Use the two-stage op amp circuit shown below to convert the photodiode current to a voltage.<br />
<br />
<center>[[Image:Part1_TransimpedanceAmplifier.jpg|700 px|thumb|center|Multi-stage photodiode amplification circuit]]</center><br />
<br />
You will have to decide how to distribute the gain between the two op amp stages in the circuit. It is best to put as much gain in early stages of a multi-stage amplifier as possible. However, when the gain of a single stage becomes very high, nonidealities of the op amp become bothersome. The first stage of the amplifier will behave reasonably well as long as the gain is less than about 10<sup>6</sup> &Omega;.<br />
<br />
# '''Analyze the photodiode amplifier circuit. Choose resistor values to achieve an overall gain that will yield an output of about 5 V when the input is 500 nA.'''<br />
# Construct the circuit on your breadboard.<br />
#* Disable the power supply before you build.<br />
#* Do not yet connect the photodiode.<br />
#* Orient the LF411 op-amps so that pin 1, which is marked with a dot on the package, is at the upper left.<br />
# Connect the +/- 15 V bus strips to power the op-amps power pins.<br />
# Connect the vertical blue bus strips to the horizontal blue bus strip.<br />
<br />
=====Test connectivity and basic operation=====<br />
<br />
# Verify that all your component connections are correct and match the design.<br />
# Check that wires and components are properly seated in the breadboard and not suspiciously loose.<br />
#* You can use the ohm-meter function of the digital multi-meter to verify continuity.<br />
# Turn on the power supply and verify that +15V appears on pin 7 and -15V on pin 4.<br />
# '''Short pins 2 and 3 (the V- and V+ inputs to the op-amp) with a small wire and measure the output voltages of IC1 and IC2.'''<br />
#* With the photodiode shorted the current signal is zero, but you may still see a significant offset voltage from 50 to 100 mV at IC1 pin 6.<br />
#* Refer to [http://measure.mit.edu/~20.309/wiki/index.php?title=Real_electronics#Real_op-amps Real Electronics, real op-amps] or ask an instructor to understand and address this non-ideal behavior.<br />
<br />
==Data acquisition hardware subsystem==<br />
<br />
{{:20.309:DAQ System}}<br />
<br />
====Summary of DAQ inputs/outputs====<br />
<br />
A special connector has been prepared for you, connected as shown below. The connections and cable wire colors are also summarized [http://dl.dropbox.com/u/12957607/DAQ_Cable_Assembly_for_DNA_melting_lab.pdf in a memo].<br />
<br />
{| class="wikitable"<br />
|-<br />
! Signal Name<br />
! Signal Location<br />
! Ground Location<br />
! Pin wire color<br />
|-<br />
! DAQ Inputs <br />
|-<br />
! RTD<br />
| AI0+<br />
| AI0-<br />
| +Orange / -Black [lone pair, not with third (red) wire]<br />
|-<br />
! Photodiode <br />
| AI1+ <br />
| AI1-<br />
| +Green / -White<br />
|}<br />
<br />
<center>[[Image:DAQ_Wire_Colors.png|450 px|thumb|center|DAQ Connection Cable]]</center><br />
<br />
==DNAMelter software==<br />
<br />
[[Image:BasicDNAMelterIcon.png|75 px|left]]<br />
Use the <code>Basic DNA Melter GUI</code> program (located on the lab computer desktop) to collect data from the apparatus.<br />
<br />
At the end of an experimental run, use the "Save" button to write the date to a file. The data will be tab-delimited and can be read into Matlab with the <code>load</code> command.<br />
<br />
Documentation for the DNAMelter software is available here: [[DNA Melting: Using the Basic DNAMelter GUI]]<br />
<br />
====If you need to debug the DAQ (skip otherwise!)====<br />
* When debugging problems, it is frequently useful to ascertain whether the data acquisition system is working properly. National Instruments provides an application for this purpose called Measurement and Automation Explorer. Use Measurement and Automation Explorer to verify that the DAQ is properly connected to and communicating with the computer. The program also provides the ability to manually control all of the DAQ system's inputs and outputs.<br />
** Launch Measurement and Automation Explorer by selecting Start->All Applications->National Instruments->Measurement and Automation.<br />
** In the "Configuration" tab, select "Devices and Interfaces" and then "NI-DAQMx Devices." Select "Dev1."<br />
** If Dev 1 does not show up, the problem is that computer cannot communicate with the DAQ system. Ensure that the USB cable is connected to the DAQ system. If the cable is connected and the device still does not show up, unplug the USB cable, count to five, and plug it back in. <br />
** If the DAQ shows up under a different name, disconnect the USB cable. Delete all the "NI USB-6xxx" entries and then reconnect the USB cable. Verify that one entry reappears and that it is called "Dev1." <br />
** Select the "Test Panels" tab to manually control or read signals from the DAQ.<br />
* If you run DNAMelter and there is a data acquisition error, for example, "<code>Data acquisition cannot start!</code>", open Measurement and Automation Explorer and follow the steps above. Run DNAMelter again. If it still doesn't work, grab a beer or ask an instructor for help.<br />
* The DAQ has only one analog-to-digital converter (ADC). A multiplexer circuit sequentially samples voltages from the analog inputs configured in the control software. If any of the inputs is outside the allowable range of +/-10V, all of the inputs may behave erratically. The voltages reported may look reasonable so this can be very misleading. If none of the DAQ readings make sense, ensure that all of the analog voltages are in the allowable range and reset the DAQ by cycling the power of the DAQ (use the switch on the 6341 or disconnect the USB of the 6212).<br />
* If the DAQ gets short-circuited, it may crash. If this happens, Matlab will frequently give a deceptive error message, for example claiming that the limits on the band-pass filter do not satisfy some condition. In this case, correct the connection and cycle the DAQ power.<br />
<br />
==Part 1 measurements==<br />
<br />
Please read this entire section before beginning. Then start at Testing the instrument, with reference back to the sample sections as-needed.<br />
<br />
===Making samples===<br />
<br />
{{Template:Biohazard warning|message=LC Green in DMSO is readily absorbed through skin. Synthetic oligonucleotides may be harmful by inhalation, ingestion, or skin absorption. Wear gloves when handling samples. Wear safety goggles at all times when pipetting the LC Green/DNA samples. Do not create aerosols. The health effects of LC Green have not been thoroughly investigated. See the LC Green and synthetic oligonucleotide under <code>../EHS Guidelines/MSDS Repository</code> in the course locker for more information.}}<br />
<br />
====Sample prep steps====<br />
<br />
#Pipet 500 &mu;L of DNA plus dye solution into a glass vial. <br />
#Pipet up to 200 &mu;L of mineral oil on top of the sample to help prevent evaporation. It will help to pipette into the corners first. Be careful not to get oil on the cuvette sides far above the sample. It will run down during your experiment and cause shifts in the photodiode signal. Place the cap loosely on the vial. <br />
#*Keep the sample vertical to make sure the oil stays on top. <br />
<br />
* You can use the same sample for several heating/cooling cycles.<br />
* Only discard a sample if you lose significant volume due to evaporation or if your signal gets too low. <br />
<br />
To clean your glass vial between samples, flush with alcohol in the waste container and rinse with water at the drain. Suck out residual liquid with the vacuum and drawn Pasteur pipette to the left of the sink.<br />
<br />
====Sample disposal====<br />
<br />
{{Template:Environmental Warning|message=Discard pipette tips with DNA sample residue in the pipette tips or the ''Biohazard Sharps'' container. Do not pour synthetic oligonucleotides with LC Green down the drain. Pour your used samples into the waste container provided in the middle of the wet bench.}}<br />
<br />
===Testing the instrument===<br />
<br />
Once your apparatus is built, use samples of fluorescein (~ 30&mu;M concentration) as a stand-in for LC Green + DNA to test the instrument. Fluorescein is cheap, non-toxic and will not bleach as quickly as LC Green.<br />
<br />
====Making a simple measurement of your instrument's low frequency SNR====<br />
<br />
Carry out a simple estimate of the signal to noise ratio (SNR) of the instrument by comparing the difference between the average signal with a sample present and with water as a null reference. This process requires a vial containing 500 uL of 30 uM fluorescein and a vial containing the same amount of water. <br />
<br />
# Run the <code>Basic DNA Melter GUI</code>. <br />
# Place a vial of DI water in your instrument.<br />
# Clear the data and wait 10 seconds.<br />
# Replace the water vial with the DNA of fluorescein vial, record for another 10 seconds, then save the data. <br />
#* Be sure that all other conditions, such as temperature, are stable throughout the test. <br />
# Segment your data into that when the water was present and that when the fluorescein was present. Calculate the average signal in each case. Record the difference between the two averages as your signal. <br />
# Now discard the water portion of your signal and subtract the average fluorescein signal value from the fluorescein signal.<br />
# Measure the standard deviation of this residual and record as record as your estimate of the noise level.<br />
# '''Calculate the SNR as signal divided by noise and record in your lab report.'''<br />
<br />
===Experiment steps===<br />
<br />
Once the instrument is confirmed to be functioning properly, follow these instructions to generate melting curve data for the 20 bp DNA-LC Green solution.<br />
<br />
#Open and run the <code>Basic DNA Melter GUI</code> in Matlab and follow the instructions there.<br />
# Confirm that block temperature is near room temperature when the Diablotek supply is off.<br />
# Apply heat to the block by switching the Diablotek on to increase the block temperature to 95&deg;C, toggle the Diablotek power in order to hold the temperature long enough for the fluorescence curve to reach a minimum, then allow the block to cool down by switching off the Diablotek.<br />
#* Other approaches may be used. One goal would be to heat the block more slowly, possibly by toggling the current while heating.<br />
# Now prepare a sample using the stock of "beater" DNA.<br />
# Place the vial in the instrument, but keep the LED off until ready to measure the melting curve.<br />
# Measure melting curves of the beater DNA sample as-desired to understand instrument operation.<br />
# Prepare a sample using fresh 20 bp 100 mM KCl DNA.<br />
# Measure a melting curve of the fresh sample.<br />
#* Be sure to save a copy in a local directory.<br />
<br />
{{:DNA Melting Report Requirements for Part 1}}<br />
<br />
==Lab manual sections==<br />
<br />
*[[Lab Manual:Measuring DNA Melting Curves]]<br />
*[[DNA Melting: Simulating DNA Melting - Basics]]<br />
*[[DNA Melting Part 1: Measuring Temperature and Fluorescence]]<br />
*[[DNA Melting Report Requirements for Part 1]]<br />
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]<br />
*[[DNA Melting Report Requirements for Part 2]]<br />
<br />
==References==<br />
<references/><br />
<br />
==Subset of datasheets==<br />
(Many more can be found online or on the course share)<br />
#[http://www.ni.com/pdf/manuals/371931f.pdf National Instruments USB-6212 user manual]<br />
#[http://www.ni.com/pdf/manuals/370784d.pdf National Instruments USB-6341 user manual]<br />
#[http://www.national.com/ds/LF/LF411.pdf Op-amp datasheet]<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/DNA_Melting_Part_1:_Measuring_Temperature_and_FluorescenceDNA Melting Part 1: Measuring Temperature and Fluorescence2015-11-08T18:38:35Z<p>Steven Nagle: /* Assembly instructions */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
<br />
[[Image:DNA Melting Block Diagram.jpg|frame|DNA melting apparatus block diagram showing the five functional groups of the instrument.]]<br />
<br />
<br />
''In theory, there is no difference between theory and practice. But, in practice, there is.'' <br />
<br />
''&mdash;[http://en.wikiquote.org/wiki/Jan_L._A._van_de_Snepscheut Jan L. A. van de Snepscheut]/[http://en.wikiquote.org/wiki/Yogi_Berra Yogi Berra]/and or [http://c2.com/cgi/wiki?DifferenceBetweenTheoryAndPractice Chuck Reid]'' <br />
<br />
You will construct a basic DNA melting instrument consisting of the five functional groups shown in the block diagram:<br />
* excitation<br />
* heating<br />
* fluorescent detection<br />
* temperature measurement<br />
* data acquisition and control<br />
<br />
A metal block will hold a sample vial in the excitation light path and transfer heat to the sample. The sample contains a combination of DNA, SYBR Green I and a salt. Recall that SYBR Green I fluoresces when it is bound to double stranded DNA (dsDNA) by a factor of up to 1000x compared to when it is bound to single stranded DNA (ssDNA). Detection of this fluorescence provides a relative measure of the fraction of dsDNA to total DNA, which can be compared to predictions from a thermodynamic model.<br />
<br />
The dye has a peak sensitivity to blue light at 497 nm so a blue LED will be used to illuminate the sample. The dye emits green light with an emission peak at 520 nm. A photodiode will be placed at 90&deg; to the LED source to detect the green light emitted by SYBR Green I. Because LEDs have a fairly broad spectrum, first a moderately narrow band-pass filter will be used to limit the excitation wavelength to between 450 and 490 nm. Then a complimentary long-pass emission filter will be placed between the sample and detector to pass only wavelengths longer than 515 nm. This filter combination, when used with this dye system, eliminates any background signal due to the excitation light.<br />
<br />
The sample block will be heated by a TEC (thermoelectric cooler) and its temperature measured by an embedded RTD (resistance temperature detector). Also, since photodiodes produce a very small amount of current, a high gain amplifier will convert the photodiode current into a measurable voltage. The DAQ (data acquisition card) and PC will digitize the amplified photodiode and RTD signals. A Matlab program is provided to record these fluorescence and temperature signals over time and save the data to a file. The file can then be loaded into Matlab for analysis, or if you prefer, Python or equivalent where the signals will be converted into dsDNA concentration and sample temperature. Finally, the dsDNA sample melting temperature will be estimated from a plot of df/dT vs T (''f'' represents the fraction of total DNA that is in double-stranded form) and from a comparison to the thermodynamic model.<br />
<br />
The functional groups in the block diagram are one of two fundamental frameworks used to describe an instrument. The other sub-divides the instrument into categories that often mirror historical areas of engineering. The former is focused on what will be done, including the overall scientific goals and the physical processes to be measured. The latter is focused on how it will be done, e.g. using optics, electronics or perhaps a practical knowledge of thermal management. A good engineer will constantly be thinking of the instrument in both frameworks. <br />
<br />
==Mechanical and thermal components==<br />
<br />
[[Image:DNA_Setup_v2_Iso.png|600 px|thumb|center|DNA Melting System Model]]<br />
<br />
Mechanical support for the heating block, sample and optics will be built on a portable optical breadboard from [http://www.thorlabs.com ThorLabs]. Most of the components in the lab are from this supplier because the selection is large, the quality is quite good and the price reasonable. Although you are free to modify the design as the lab progresses, begin a build from the standard design shown above and demonstrated by the example in the lab. Gather the following Thorlabs components but do not start to assemble.<br />
<br />
* 1 MB1224 optical breadboard<br />
* 2 CL5 clamps (or similar)<br />
* 2 1/4-20 5/8" screws<br />
* 4 1/4-20 1/4" screws<br />
<br />
The thermal subsystem includes all components to control heat flow into and out of the sample and monitor its temperature. Heating is accomplished using thermal electric coolers (TECs) in reverse. The hot side is placed in direct contact with the heating block, which is in direct contact with the sample vial. The TECs will be powered by manually controlling the Diablotek power supply. To avoid contamination of the sample or perturbation of its temperature, the temperature of a resistive temperature detector (RTD) embedded in the heating block will be monitored as a proxy for the sample temperature. In this part of the lab, air convection will cool down the sample when the Diablotek is switched off. The heat sink will help and the fan may be connected to a triple-output power supply to help, but these are included primarily for use in Part 2.<br />
<br />
===Parts===<br />
Gather the following components from the bins on the Center Cabinets, the DNA Melting drawer at the wet bench or on the East Cabinets:<br />
<br />
[[Image:CenterCabinets.jpg|right|250px|thumb|'''Center Cabinets''' The bins for most parts that you need will be set out on top of these cabinets, roughly in the center of the lab.]]<br />
[[Image:WetBench.jpg|right|250px|thumb|'''Wet Bench''' Supplies for our wet bench can be found in the three drawers at the left of the bench, the drawer left of the sink and on the wall behind and to the left as you face the wet bench. The drawer at the right of the wet bench contains various supplies for the DNA Melting instrument.]]<br />
[[Image:EastCabinets.jpg|right|250px|thumb|'''East Cabinets''' The center shelves of the East Cabinets contain the electronic breadboards, breadboard jumper wire, TEC connectors and TEC power supplies. Often these supplies are laid out on top of these cabinets.]]<br />
<br />
* These components are already assembled for you: A metal support bracket, having a fan, heatsink, 2 TEC heaters/coolers, and a heating block attached to it<br />
* 3 wire nuts<br />
* 1 Diablotek 250 W power supply (at your station or on a shelf above your station)<br />
* 1 red/black TEC extension wire assembly (in a box on the ''''Center Cabinets'''')<br />
<br />
===Assembly instructions===<br />
<br />
[[Image:TEC_picture.jpg|150 px|thumb|right|TEC showing hot side up when current flows into the red lead]]<br />
<br />
Ensure that the following have been done/attached to your metal bracket:<br />
# Attach the fan on the side of the support bracket using the 6-32 screws and nuts.<br />
# Attach the 2x2 heat sink to the bottom of the support bracket using two 4-40 screws at opposite corners.<br />
# Attach the support bracket to the MB1224 breadboard using 1/4-20 1/4" screws.<br />
# Now place the TECs on a napkin or two, with the red lead extending down and to the ''left'' as shown in the figure.<br />
# Apply a very thin layer of thermal grease to the exposed side of one of the TECs using a wooden stick from the drawer.<br />
#* Use a small amount of grease. Too much is worse than none.<br />
#* Best practice is to apply a baby-pea-sized amount, spread with the stick, smooth and remove excess almost to the point of total removal.<br />
# Now place the second TEC on top of the first.<br />
#* If the two TECs slide freely against each other, then there is too much grease.<br />
#** Separate the TECs and smooth/remove the grease until a modest force is required to slide them.<br />
#* Verify that the colors of the leads match. If not, flip one TEC.<br />
# Connect the black lead of the top TEC to the red lead of the bottom TEC using a wire nut.<br />
# Connect the TEC to the extension wire assembly using a wire nut to join the white wire to the red lead of the top TEC and the black wire to the black lead on the bottom TEC.<br />
# Join the white connector on the assembly to the black 2x2 connector of the Diabloteck.<br />
# Test the TECs by holding the them between thumb and finger while briefly flipping the switch on the Diablotek.<br />
#* One side should immediately heat up while the other immediately cools.<br />
#* The top side should be the hot side when the red wires extend down and to the '''left'''.<br />
#* If the Diablotek immediately shuts off (the fan will stop spinning) then there is a short. Check all connections.<br />
# Once the TEC assembly checks out, butter the bottom side of the assembly and place that side on the heat sink with the leads facing to the right.<br />
# Finally, apply thermal grease to the top side of the TEC assembly, place the heating block on top and anchor to the heat sink using the 1/2" long 4-40 screws and plastic washers.<br />
<br />
==Optical subsystem==<br />
<br />
A good optical subsystem design will be compact, stable and simple. It includes the optical components of the excitation and fluorescence detection functional groups from the block diagram. A good design will efficiently illuminate the sample and collect the maximum possible amount of emitted fluorescent light. There are a variety of ways to construct the apparatus. Use the model above and the example system in the lab as guides.<br />
<br />
The following is a rough set of instructions if you're not clear on how to replicate the example system in the lab. In addition to the following parts, you might want to add a few lenses to collimate your LED onto your sample, and to focus the light coming from your sample onto your photodiode. <br />
<br />
* 2 PH2 post holders<br />
* 2 BA1 post holder bases<br />
* 2 1/4-20 1/4" screws<br />
* 2 1/4-20 5/8" screws<br />
* 2 1/4" washers<br />
* 4 CP02 cage plates<br />
* 2 8-32 1/2" setscrews<br />
* 2 TR2 posts<br />
* 6 ER3 cage rails<br />
* 12 4-40 1/4" setscrews (or similar length)<br />
* 4 SML05 lens tubes<br />
* 4 SM1LTRR retaining rings<br />
* 1 blue LED PCB<br />
* 1 D470/40 excitation filter<br />
* 1 E515LPv2 emission filter<br />
* 1 SM1A6 adapter between SM1 and SM05 threads<br />
* 1 SM05PD1A mounted photodiode<br />
<br />
===Assembly instructions===<br />
Use an Instructor instrument or the Solid Works model above as guide to assemble your optical subassembly. Note also that these instructions are also a guid. If you wish to build your own personal design, please do.<br />
<br />
# Attach each PH2 to a BA1 using 1/4" screws.<br />
# Attach two of the CP02 cage plates to two TR2 posts using an 8-32 setscrew.<br />
# Insert the TR2/CP02 assembly into the PH2/BA1 assemblies and lock in place with the TS25H setscrews.<br />
# Insert three ER4 cage rails into each CP02 above and lock in place with SS4S025 set screws or similar.<br />
#* Leave the blank hole on the bottom of the assembly.<br />
# Attach one of the remaining CP02 cage plates on the free end of each assembly above and lock in place.<br />
# Attach each of the above assemblies to the MB1224 using a washer and 1/4-20 screw less than 1" in length.<br />
# Arrange on the MB1224 and adjust CP02 heights as desired.<br />
# Mount the LED PCB in an SML05 lens tube and fix with a retaining ring.<br />
# Mount the excitation and emission filters in each of two lens tubes and fix with retaining ring.<br />
#* The arrow label on the thickness of the filter points ''toward the light source''.<br />
# Thread the SM1A6 adapter into a lens tube and fix with a retaining ring.<br />
# Thread the photodiode into the SM1A6 adapter.<br />
#* Teflon pipe thread tape can be used to hold the photodiode in the desired position.<br />
# Mount each lens tube in the appropriate CP02 cage plates.<br />
# Arrange each the resultant assemblies on the breadboard to most fully illuminate the sample vial and to gather the most of the resultant light on the photodiode.<br />
<br />
==Electronics subsystem==<br />
<br />
The electrical subsystem includes the LED with a current-limiting resistor, the photodiode, the transimpedance amplifier and a temperature detection circuit. The remainder of this section provides an example of each of these systems and instructions to build and test each one. Nevertheless, alternate ideas are welcome. Feel free to discuss with the Instructors.<br />
<br />
===Parts===<br />
<br />
Gather the following components from the electronics drawer to the right of the optics drawers, from the counter top opposite the wet bench and from the cable organizer on the East wall to the right of the white board.<br />
* 1 electronic breadboard<br />
* 1 jump wire kit<br />
* 2 red 3' banana cable or similar<br />
* 1 black 3' banana cable or similar<br />
* individual wire, as-needed from the scrap wire box or the wire spools opposite the wet bench<br />
* 1 62 &Omega; resistor from the center counter top on the South wall<br />
* 1 15 k&Omega; resistor<br />
* other resistors, as-needed for the amplifier<br />
* 1 blue LED from the DNA Melting drawer at the far right of the wet bench<br />
* 2 LF411 op-amps from the electronics drawer<br />
* 4 capacitors between 0.1 and 22 uF from either the electronics drawer or the counter top where the screws are stored.<br />
<br />
===Assembly instructions===<br />
<br />
====LED circuit====<br />
The led circuit consists of a 62 &Omega; resistor in series with a blue, jumbo LED. The circuit will be powered by the 5 volt fixed output of the lab power supply. The 62 &Omega; resistor prevents excessive current from flowing through the LED. Excessive current will let the smoke out of the LED.<br />
<br />
# Build the LED circuit on your electronic breadboard.<br />
#* Don't forget to connect the resistor. Directly connecting the LED to 5 V will blow out the LED.<br />
# Disable the output of the lab power supply.<br />
# Connect the LED circuit to the fixed 5 V output.<br />
<br />
====Temperature detection circuit====<br />
A platinum RTD is embedded inside the heating block. The resistance of the RTD varies with temperature according to the equation ''R<sub>RTD</sub>''=1000 &Omega; + 3.85 ''&theta;'', where ''&theta;'' is the temperature of the RTD in degrees Celsius. The easiest way to make a temperature measurement is to connect the RTD in series with a fixed resistor and a power supply. The output voltage of the resulting divider circuit varies as a function of temperature. <br />
<br />
The maximum current of the RTD is 1 mA. You will use a 15 V supply to the circuit, so the resistor must be at least 14 k&Omega;. Use a 15 k&Omega; resistor.<br />
<br />
# '''Find an equation for ''V<sub>out</sub>'' as a function of temperature. What voltage do you expect given an input voltage of 15 V with the RTD at room temperature.'''<br />
# Turn off the output of the triple-output power supply.<br />
# Connect the RTD to the electronic breadboard.<br />
# '''Measure the resistance of the 15 k&Omega; resistor with a DMM and record the value in your lab report.'''<br />
# Connect the 15k&Omega; resistor in series with the RTD.<br />
# Set the power supply for '''SERIES''' operation.<br />
# Set the output voltage to 15 V on the supply and make note of its exact value using the DMM.<br />
# Set the current limit to 0.1 A.<br />
# Connect the positive terminal of '''CH1''' to a banana terminal on the breadboard for +15 V and wire it to the top red bus strip for +15 V supply.<br />
# Connect the negative terminal of '''CH2''' to a banana terminal on the breadboard for -15 V.<br />
# Connect the positive terminal of '''CH2''' to the black terminal on the breadboard for a ground reference and wire it to the top blue bus strip.<br />
#* Use three jump wires to minimize the parasitic voltage drop due to the breadboard tie points.<br />
# Connect the +15 V bus strip to the second vertical, red bus strip and wire this bus strip to the top of your 15 k&Omega; resistor.<br />
# Turn on the output of the power supply and measure the voltage across the RTD using the DMM.<br />
# '''What is the measured voltage of the RTD at room temperature and how does it compare to your predictions above?'''<br />
# '''What is the temperature, based on your measured voltage and how does it compare with the clock on the wall (or some better measure)?'''<br />
# '''How is the temperature calculation affected by a 1% change in the 15 V input voltage?'''<br />
<br />
====Photodiode amplifier circuit====<br />
<br />
The current produced by the photodiode detector is a few tens of nanoamperes. This very small current must be amplified to a level where it can be easily measured. The data acquisition system you will use can measure voltages between + and -10 V. Use the two-stage op amp circuit shown below to convert the photodiode current to a voltage.<br />
<br />
<center>[[Image:Part1_TransimpedanceAmplifier.jpg|700 px|thumb|center|Multi-stage photodiode amplification circuit]]</center><br />
<br />
You will have to decide how to distribute the gain between the two op amp stages in the circuit. It is best to put as much gain in early stages of a multi-stage amplifier as possible. However, when the gain of a single stage becomes very high, nonidealities of the op amp become bothersome. The first stage of the amplifier will behave reasonably well as long as the gain is less than about 10<sup>6</sup> &Omega;.<br />
<br />
# '''Analyze the photodiode amplifier circuit. Choose resistor values to achieve an overall gain that will yield an output of about 5 V when the input is 500 nA.'''<br />
# Construct the circuit on your breadboard.<br />
#* Disable the power supply before you build.<br />
#* Do not yet connect the photodiode.<br />
#* Orient the LF411 op-amps so that pin 1, which is marked with a dot on the package, is at the upper left.<br />
# Connect the +/- 15 V bus strips to power the op-amps power pins.<br />
# Connect the vertical blue bus strips to the horizontal blue bus strip.<br />
<br />
=====Test connectivity and basic operation=====<br />
<br />
# Verify that all your component connections are correct and match the design.<br />
# Check that wires and components are properly seated in the breadboard and not suspiciously loose.<br />
#* You can use the ohm-meter function of the digital multi-meter to verify continuity.<br />
# Turn on the power supply and verify that +15V appears on pin 7 and -15V on pin 4.<br />
# '''Short pins 2 and 3 (the V- and V+ inputs to the op-amp) with a small wire and measure the output voltages of IC1 and IC2.'''<br />
#* With the photodiode shorted the current signal is zero, but you may still see a significant offset voltage from 50 to 100 mV at IC1 pin 6.<br />
#* Refer to [http://measure.mit.edu/~20.309/wiki/index.php?title=Real_electronics#Real_op-amps Real Electronics, real op-amps] or ask an instructor to understand and address this non-ideal behavior.<br />
<br />
==Data acquisition hardware subsystem==<br />
<br />
{{:20.309:DAQ System}}<br />
<br />
====Summary of DAQ inputs/outputs====<br />
<br />
A special connector has been prepared for you, connected as shown below. The connections and cable wire colors are also summarized [http://dl.dropbox.com/u/12957607/DAQ_Cable_Assembly_for_DNA_melting_lab.pdf in a memo].<br />
<br />
{| class="wikitable"<br />
|-<br />
! Signal Name<br />
! Signal Location<br />
! Ground Location<br />
! Pin wire color<br />
|-<br />
! DAQ Inputs <br />
|-<br />
! RTD<br />
| AI0+<br />
| AI0-<br />
| +Orange / -Black [lone pair, not with third (red) wire]<br />
|-<br />
! Photodiode <br />
| AI1+ <br />
| AI1-<br />
| +Green / -White<br />
|}<br />
<br />
<center>[[Image:DAQ_Wire_Colors.png|450 px|thumb|center|DAQ Connection Cable]]</center><br />
<br />
==DNAMelter software==<br />
<br />
[[Image:BasicDNAMelterIcon.png|75 px|left]]<br />
Use the <code>Basic DNA Melter GUI</code> program (located on the lab computer desktop) to collect data from the apparatus.<br />
<br />
At the end of an experimental run, use the "Save" button to write the date to a file. The data will be tab-delimited and can be read into Matlab with the <code>load</code> command.<br />
<br />
Documentation for the DNAMelter software is available here: [[DNA Melting: Using the Basic DNAMelter GUI]]<br />
<br />
====If you need to debug the DAQ (skip otherwise!)====<br />
* When debugging problems, it is frequently useful to ascertain whether the data acquisition system is working properly. National Instruments provides an application for this purpose called Measurement and Automation Explorer. Use Measurement and Automation Explorer to verify that the DAQ is properly connected to and communicating with the computer. The program also provides the ability to manually control all of the DAQ system's inputs and outputs.<br />
** Launch Measurement and Automation Explorer by selecting Start->All Applications->National Instruments->Measurement and Automation.<br />
** In the "Configuration" tab, select "Devices and Interfaces" and then "NI-DAQMx Devices." Select "Dev1."<br />
** If Dev 1 does not show up, the problem is that computer cannot communicate with the DAQ system. Ensure that the USB cable is connected to the DAQ system. If the cable is connected and the device still does not show up, unplug the USB cable, count to five, and plug it back in. <br />
** If the DAQ shows up under a different name, disconnect the USB cable. Delete all the "NI USB-6xxx" entries and then reconnect the USB cable. Verify that one entry reappears and that it is called "Dev1." <br />
** Select the "Test Panels" tab to manually control or read signals from the DAQ.<br />
* If you run DNAMelter and there is a data acquisition error, for example, "<code>Data acquisition cannot start!</code>", open Measurement and Automation Explorer and follow the steps above. Run DNAMelter again. If it still doesn't work, grab a beer or ask an instructor for help.<br />
* The DAQ has only one analog-to-digital converter (ADC). A multiplexer circuit sequentially samples voltages from the analog inputs configured in the control software. If any of the inputs is outside the allowable range of +/-10V, all of the inputs may behave erratically. The voltages reported may look reasonable so this can be very misleading. If none of the DAQ readings make sense, ensure that all of the analog voltages are in the allowable range and reset the DAQ by cycling the power of the DAQ (use the switch on the 6341 or disconnect the USB of the 6212).<br />
* If the DAQ gets short-circuited, it may crash. If this happens, Matlab will frequently give a deceptive error message, for example claiming that the limits on the band-pass filter do not satisfy some condition. In this case, correct the connection and cycle the DAQ power.<br />
<br />
==Part 1 measurements==<br />
<br />
Please read this entire section before beginning. Then start at Testing the instrument, with reference back to the sample sections as-needed.<br />
<br />
===Making samples===<br />
<br />
{{Template:Biohazard warning|message=LC Green in DMSO is readily absorbed through skin. Synthetic oligonucleotides may be harmful by inhalation, ingestion, or skin absorption. Wear gloves when handling samples. Wear safety goggles at all times when pipetting the LC Green/DNA samples. Do not create aerosols. The health effects of LC Green have not been thoroughly investigated. See the LC Green and synthetic oligonucleotide under <code>../EHS Guidelines/MSDS Repository</code> in the course locker for more information.}}<br />
<br />
====Sample prep steps====<br />
<br />
#Pipet 500 &mu;L of DNA plus dye solution into a glass vial. <br />
#Pipet up to 200 &mu;L of mineral oil on top of the sample to help prevent evaporation. It will help to pipette into the corners first. Be careful not to get oil on the cuvette sides far above the sample. It will run down during your experiment and cause shifts in the photodiode signal. Place the cap loosely on the vial. <br />
#*Keep the sample vertical to make sure the oil stays on top. <br />
<br />
* You can use the same sample for several heating/cooling cycles.<br />
* Only discard a sample if you lose significant volume due to evaporation or if your signal gets too low. <br />
<br />
To clean your glass vial between samples, flush with alcohol in the waste container and rinse with water at the drain. Suck out residual liquid with the vacuum and drawn Pasteur pipette to the left of the sink.<br />
<br />
====Sample disposal====<br />
<br />
{{Template:Environmental Warning|message=Discard pipette tips with DNA sample residue in the pipette tips or the ''Biohazard Sharps'' container. Do not pour synthetic oligonucleotides with LC Green down the drain. Pour your used samples into the waste container provided in the middle of the wet bench.}}<br />
<br />
===Testing the instrument===<br />
<br />
Once your apparatus is built, use samples of fluorescein (~ 30&mu;M concentration) as a stand-in for LC Green + DNA to test the instrument. Fluorescein is cheap, non-toxic and will not bleach as quickly as LC Green.<br />
<br />
====Making a simple measurement of your instrument's low frequency SNR====<br />
<br />
Carry out a simple estimate of the signal to noise ratio (SNR) of the instrument by comparing the difference between the average signal with a sample present and with water as a null reference. This process requires a vial containing 500 uL of 30 uM fluorescein and a vial containing the same amount of water. <br />
<br />
# Run the <code>Basic DNA Melter GUI</code>. <br />
# Place a vial of DI water in your instrument.<br />
# Clear the data and wait 10 seconds.<br />
# Replace the water vial with the DNA of fluorescein vial, record for another 10 seconds, then save the data. <br />
#* Be sure that all other conditions, such as temperature, are stable throughout the test. <br />
# Segment your data into that when the water was present and that when the fluorescein was present. Calculate the average signal in each case. Record the difference between the two averages as your signal. <br />
# Now discard the water portion of your signal and subtract the average fluorescein signal value from the fluorescein signal.<br />
# Measure the standard deviation of this residual and record as record as your estimate of the noise level.<br />
# '''Calculate the SNR as signal divided by noise and record in your lab report.'''<br />
<br />
===Experiment steps===<br />
<br />
Once the instrument is confirmed to be functioning properly, follow these instructions to generate melting curve data for the 20 bp DNA-LC Green solution.<br />
<br />
#Open and run the <code>Basic DNA Melter GUI</code> in Matlab and follow the instructions there.<br />
# Confirm that block temperature is near room temperature when the Diablotek supply is off.<br />
# Apply heat to the block by switching the Diablotek on to increase the block temperature to 95&deg;C, toggle the Diablotek power in order to hold the temperature long enough for the fluorescence curve to reach a minimum, then allow the block to cool down by switching off the Diablotek.<br />
#* Other approaches may be used. One goal would be to heat the block more slowly, possibly by toggling the current while heating.<br />
# Now prepare a sample using the stock of "beater" DNA.<br />
# Place the vial in the instrument, but keep the LED off until ready to measure the melting curve.<br />
# Measure melting curves of the beater DNA sample as-desired to understand instrument operation.<br />
# Prepare a sample using fresh 20 bp 100 mM KCl DNA.<br />
# Measure a melting curve of the fresh sample.<br />
#* Be sure to save a copy in a local directory.<br />
<br />
{{:DNA Melting Report Requirements for Part 1}}<br />
<br />
==Lab manual sections==<br />
<br />
*[[Lab Manual:Measuring DNA Melting Curves]]<br />
*[[DNA Melting: Simulating DNA Melting - Basics]]<br />
*[[DNA Melting Part 1: Measuring Temperature and Fluorescence]]<br />
*[[DNA Melting Report Requirements for Part 1]]<br />
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]<br />
*[[DNA Melting Report Requirements for Part 2]]<br />
<br />
==References==<br />
<references/><br />
<br />
==Subset of datasheets==<br />
(Many more can be found online or on the course share)<br />
#[http://www.ni.com/pdf/manuals/371931f.pdf National Instruments USB-6212 user manual]<br />
#[http://www.ni.com/pdf/manuals/370784d.pdf National Instruments USB-6341 user manual]<br />
#[http://www.national.com/ds/LF/LF411.pdf Op-amp datasheet]<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/DNA_Melting_Part_1:_Measuring_Temperature_and_FluorescenceDNA Melting Part 1: Measuring Temperature and Fluorescence2015-11-08T18:36:02Z<p>Steven Nagle: </p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
<br />
[[Image:DNA Melting Block Diagram.jpg|frame|DNA melting apparatus block diagram showing the five functional groups of the instrument.]]<br />
<br />
<br />
''In theory, there is no difference between theory and practice. But, in practice, there is.'' <br />
<br />
''&mdash;[http://en.wikiquote.org/wiki/Jan_L._A._van_de_Snepscheut Jan L. A. van de Snepscheut]/[http://en.wikiquote.org/wiki/Yogi_Berra Yogi Berra]/and or [http://c2.com/cgi/wiki?DifferenceBetweenTheoryAndPractice Chuck Reid]'' <br />
<br />
You will construct a basic DNA melting instrument consisting of the five functional groups shown in the block diagram:<br />
* excitation<br />
* heating<br />
* fluorescent detection<br />
* temperature measurement<br />
* data acquisition and control<br />
<br />
A metal block will hold a sample vial in the excitation light path and transfer heat to the sample. The sample contains a combination of DNA, SYBR Green I and a salt. Recall that SYBR Green I fluoresces when it is bound to double stranded DNA (dsDNA) by a factor of up to 1000x compared to when it is bound to single stranded DNA (ssDNA). Detection of this fluorescence provides a relative measure of the fraction of dsDNA to total DNA, which can be compared to predictions from a thermodynamic model.<br />
<br />
The dye has a peak sensitivity to blue light at 497 nm so a blue LED will be used to illuminate the sample. The dye emits green light with an emission peak at 520 nm. A photodiode will be placed at 90&deg; to the LED source to detect the green light emitted by SYBR Green I. Because LEDs have a fairly broad spectrum, first a moderately narrow band-pass filter will be used to limit the excitation wavelength to between 450 and 490 nm. Then a complimentary long-pass emission filter will be placed between the sample and detector to pass only wavelengths longer than 515 nm. This filter combination, when used with this dye system, eliminates any background signal due to the excitation light.<br />
<br />
The sample block will be heated by a TEC (thermoelectric cooler) and its temperature measured by an embedded RTD (resistance temperature detector). Also, since photodiodes produce a very small amount of current, a high gain amplifier will convert the photodiode current into a measurable voltage. The DAQ (data acquisition card) and PC will digitize the amplified photodiode and RTD signals. A Matlab program is provided to record these fluorescence and temperature signals over time and save the data to a file. The file can then be loaded into Matlab for analysis, or if you prefer, Python or equivalent where the signals will be converted into dsDNA concentration and sample temperature. Finally, the dsDNA sample melting temperature will be estimated from a plot of df/dT vs T (''f'' represents the fraction of total DNA that is in double-stranded form) and from a comparison to the thermodynamic model.<br />
<br />
The functional groups in the block diagram are one of two fundamental frameworks used to describe an instrument. The other sub-divides the instrument into categories that often mirror historical areas of engineering. The former is focused on what will be done, including the overall scientific goals and the physical processes to be measured. The latter is focused on how it will be done, e.g. using optics, electronics or perhaps a practical knowledge of thermal management. A good engineer will constantly be thinking of the instrument in both frameworks. <br />
<br />
==Mechanical and thermal components==<br />
<br />
[[Image:DNA_Setup_v2_Iso.png|600 px|thumb|center|DNA Melting System Model]]<br />
<br />
Mechanical support for the heating block, sample and optics will be built on a portable optical breadboard from [http://www.thorlabs.com ThorLabs]. Most of the components in the lab are from this supplier because the selection is large, the quality is quite good and the price reasonable. Although you are free to modify the design as the lab progresses, begin a build from the standard design shown above and demonstrated by the example in the lab. Gather the following Thorlabs components but do not start to assemble.<br />
<br />
* 1 MB1224 optical breadboard<br />
* 2 CL5 clamps (or similar)<br />
* 2 1/4-20 5/8" screws<br />
* 4 1/4-20 1/4" screws<br />
<br />
The thermal subsystem includes all components to control heat flow into and out of the sample and monitor its temperature. Heating is accomplished using thermal electric coolers (TECs) in reverse. The hot side is placed in direct contact with the heating block, which is in direct contact with the sample vial. The TECs will be powered by manually controlling the Diablotek power supply. To avoid contamination of the sample or perturbation of its temperature, the temperature of a resistive temperature detector (RTD) embedded in the heating block will be monitored as a proxy for the sample temperature. In this part of the lab, air convection will cool down the sample when the Diablotek is switched off. The heat sink will help and the fan may be connected to a triple-output power supply to help, but these are included primarily for use in Part 2.<br />
<br />
===Parts===<br />
Gather the following components from the bins on the Center Cabinets, the DNA Melting drawer at the wet bench or on the East Cabinets:<br />
<br />
[[Image:CenterCabinets.jpg|right|250px|thumb|'''Center Cabinets''' The bins for most parts that you need will be set out on top of these cabinets, roughly in the center of the lab.]]<br />
[[Image:WetBench.jpg|right|250px|thumb|'''Wet Bench''' Supplies for our wet bench can be found in the three drawers at the left of the bench, the drawer left of the sink and on the wall behind and to the left as you face the wet bench. The drawer at the right of the wet bench contains various supplies for the DNA Melting instrument.]]<br />
[[Image:EastCabinets.jpg|right|250px|thumb|'''East Cabinets''' The center shelves of the East Cabinets contain the electronic breadboards, breadboard jumper wire, TEC connectors and TEC power supplies. Often these supplies are laid out on top of these cabinets.]]<br />
<br />
* These components are already assembled for you: A metal support bracket, having a fan, heatsink, 2 TEC heaters/coolers, and a heating block attached to it<br />
* 3 wire nuts<br />
* 1 Diablotek 250 W power supply (at your station or on a shelf above your station)<br />
* 1 red/black TEC extension wire assembly (in a box on the ''''Center Cabinets'''')<br />
<br />
===Assembly instructions===<br />
<br />
[[Image:TEC_picture.jpg|150 px|thumb|right|TEC showing hot side up when current flows into the red lead]]<br />
<br />
Ensure that the following have been done/attached to your metal bracket:<br />
# Attach the fan on the side of the support bracket using the 6-32 screws and nuts.<br />
# Attach the 2x2 heat sink to the bottom of the support bracket using two 4-40 screws at opposite corners.<br />
# Attach the support bracket to the MB1224 breadboard using 1/4-20 1/4" screws.<br />
# Now place the TECs on a napkin or two, with the red lead extending down and to the ''left'' as shown in the figure.<br />
# Apply a very thin layer of thermal grease to the exposed side of one of the TECs using a wooden stick from the drawer.<br />
#* Use a small amount of grease. Too much is worse than none.<br />
#* Best practice is to apply a baby-pea-sized amount, spread with the stick, smooth and remove excess almost to the point of total removal.<br />
# Now place the second TEC on top of the first.<br />
#* If the two TECs slide freely against each other, then there is too much grease.<br />
#** Separate the TECs and smooth/remove the grease until a modest force is required to slide them.<br />
#* Verify that the colors of the leads match. If not, flip one TEC.<br />
# Connect the black lead of the top TEC to the red lead of the bottom TEC using a wire nut.<br />
# Connect the TEC to the extension wire assembly using a wire nut to join the white wire to the red lead of the top TEC and the black wire to the black lead on the bottom TEC.<br />
# Join the white connector on the assembly to the black 2x2 connector of the Diabloteck.<br />
# Test the TECs by holding the them between thumb and finger while briefly flipping the switch on the Diablotek.<br />
#* One side should immediately heat up while the other immediately cools.<br />
#* The top side should be the hot side when the red wires extend down and to the '''left'''.<br />
#* If the Diablotek immediately shuts off (the fan will stop spinning) then there is a short. Check all connections.<br />
# Once the TEC assembly checks out, butter the bottom side of the assembly and place that side on the heat sink with the leads facing to the right.<br />
# Finally, apply thermal grease to the top side of the TEC assembly, place the heating block on top and anchor to the heat sink using the 1/2" long 4-40 screws and plastic washers.<br />
<br />
==Optical subsystem==<br />
<br />
A good optical subsystem design will be compact, stable and simple. It includes the optical components of the excitation and fluorescence detection functional groups from the block diagram. A good design will efficiently illuminate the sample and collect the maximum possible amount of emitted fluorescent light. There are a variety of ways to construct the apparatus. Use the model above and the example system in the lab as guides.<br />
<br />
The following is a rough set of instructions if you're not clear on how to replicate the example system in the lab. In addition to the following parts, you might want to add a few lenses to collimate your LED onto your sample, and to focus the light coming from your sample onto your photodiode. <br />
<br />
* 2 PH2 post holders<br />
* 2 BA1 post holder bases<br />
* 2 1/4-20 1/4" screws<br />
* 2 1/4-20 5/8" screws<br />
* 2 1/4" washers<br />
* 4 CP02 cage plates<br />
* 2 8-32 1/2" setscrews<br />
* 2 TR2 posts<br />
* 6 ER3 cage rails<br />
* 12 4-40 1/4" setscrews (or similar length)<br />
* 4 SML05 lens tubes<br />
* 4 SM1LTRR retaining rings<br />
* 1 blue LED PCB<br />
* 1 D470/40 excitation filter<br />
* 1 E515LPv2 emission filter<br />
* 1 SM1A6 adapter between SM1 and SM05 threads<br />
* 1 SM05PD1A mounted photodiode<br />
<br />
===Assembly instructions===<br />
<br />
# Attach each PH2 to a BA1 using 1/4" screws.<br />
# Attach two of the CP02 cage plates to two TR2 posts using an 8-32 setscrew.<br />
# Insert the TR2/CP02 assembly into the PH2/BA1 assemblies and lock in place with the TS25H setscrews.<br />
# Insert three ER4 cage rails into each CP02 above and lock in place with SS4S025 set screws or similar.<br />
#* Leave the blank hole on the bottom of the assembly.<br />
# Attach one of the remaining CP02 cage plates on the free end of each assembly above and lock in place.<br />
# Attach each of the above assemblies to the MB1224 using a washer and 1/4-20 screw less than 1" in length.<br />
# Arrange on the MB1224 and adjust CP02 heights as desired.<br />
# Mount the LED PCB in an SML05 lens tube and fix with a retaining ring.<br />
# Mount the excitation and emission filters in each of two lens tubes and fix with retaining ring.<br />
#* The arrow label on the thickness of the filter points ''toward the light source''.<br />
# Thread the SM1A6 adapter into a lens tube and fix with a retaining ring.<br />
# Thread the photodiode into the SM1A6 adapter.<br />
#* Teflon pipe thread tape can be used to hold the photodiode in the desired position.<br />
# Mount each lens tube in the appropriate CP02 cage plates.<br />
# Arrange each the resultant assemblies on the breadboard to most fully illuminate the sample vial and to gather the most of the resultant light on the photodiode.<br />
<br />
==Electronics subsystem==<br />
<br />
The electrical subsystem includes the LED with a current-limiting resistor, the photodiode, the transimpedance amplifier and a temperature detection circuit. The remainder of this section provides an example of each of these systems and instructions to build and test each one. Nevertheless, alternate ideas are welcome. Feel free to discuss with the Instructors.<br />
<br />
===Parts===<br />
<br />
Gather the following components from the electronics drawer to the right of the optics drawers, from the counter top opposite the wet bench and from the cable organizer on the East wall to the right of the white board.<br />
* 1 electronic breadboard<br />
* 1 jump wire kit<br />
* 2 red 3' banana cable or similar<br />
* 1 black 3' banana cable or similar<br />
* individual wire, as-needed from the scrap wire box or the wire spools opposite the wet bench<br />
* 1 62 &Omega; resistor from the center counter top on the South wall<br />
* 1 15 k&Omega; resistor<br />
* other resistors, as-needed for the amplifier<br />
* 1 blue LED from the DNA Melting drawer at the far right of the wet bench<br />
* 2 LF411 op-amps from the electronics drawer<br />
* 4 capacitors between 0.1 and 22 uF from either the electronics drawer or the counter top where the screws are stored.<br />
<br />
===Assembly instructions===<br />
<br />
====LED circuit====<br />
The led circuit consists of a 62 &Omega; resistor in series with a blue, jumbo LED. The circuit will be powered by the 5 volt fixed output of the lab power supply. The 62 &Omega; resistor prevents excessive current from flowing through the LED. Excessive current will let the smoke out of the LED.<br />
<br />
# Build the LED circuit on your electronic breadboard.<br />
#* Don't forget to connect the resistor. Directly connecting the LED to 5 V will blow out the LED.<br />
# Disable the output of the lab power supply.<br />
# Connect the LED circuit to the fixed 5 V output.<br />
<br />
====Temperature detection circuit====<br />
A platinum RTD is embedded inside the heating block. The resistance of the RTD varies with temperature according to the equation ''R<sub>RTD</sub>''=1000 &Omega; + 3.85 ''&theta;'', where ''&theta;'' is the temperature of the RTD in degrees Celsius. The easiest way to make a temperature measurement is to connect the RTD in series with a fixed resistor and a power supply. The output voltage of the resulting divider circuit varies as a function of temperature. <br />
<br />
The maximum current of the RTD is 1 mA. You will use a 15 V supply to the circuit, so the resistor must be at least 14 k&Omega;. Use a 15 k&Omega; resistor.<br />
<br />
# '''Find an equation for ''V<sub>out</sub>'' as a function of temperature. What voltage do you expect given an input voltage of 15 V with the RTD at room temperature.'''<br />
# Turn off the output of the triple-output power supply.<br />
# Connect the RTD to the electronic breadboard.<br />
# '''Measure the resistance of the 15 k&Omega; resistor with a DMM and record the value in your lab report.'''<br />
# Connect the 15k&Omega; resistor in series with the RTD.<br />
# Set the power supply for '''SERIES''' operation.<br />
# Set the output voltage to 15 V on the supply and make note of its exact value using the DMM.<br />
# Set the current limit to 0.1 A.<br />
# Connect the positive terminal of '''CH1''' to a banana terminal on the breadboard for +15 V and wire it to the top red bus strip for +15 V supply.<br />
# Connect the negative terminal of '''CH2''' to a banana terminal on the breadboard for -15 V.<br />
# Connect the positive terminal of '''CH2''' to the black terminal on the breadboard for a ground reference and wire it to the top blue bus strip.<br />
#* Use three jump wires to minimize the parasitic voltage drop due to the breadboard tie points.<br />
# Connect the +15 V bus strip to the second vertical, red bus strip and wire this bus strip to the top of your 15 k&Omega; resistor.<br />
# Turn on the output of the power supply and measure the voltage across the RTD using the DMM.<br />
# '''What is the measured voltage of the RTD at room temperature and how does it compare to your predictions above?'''<br />
# '''What is the temperature, based on your measured voltage and how does it compare with the clock on the wall (or some better measure)?'''<br />
# '''How is the temperature calculation affected by a 1% change in the 15 V input voltage?'''<br />
<br />
====Photodiode amplifier circuit====<br />
<br />
The current produced by the photodiode detector is a few tens of nanoamperes. This very small current must be amplified to a level where it can be easily measured. The data acquisition system you will use can measure voltages between + and -10 V. Use the two-stage op amp circuit shown below to convert the photodiode current to a voltage.<br />
<br />
<center>[[Image:Part1_TransimpedanceAmplifier.jpg|700 px|thumb|center|Multi-stage photodiode amplification circuit]]</center><br />
<br />
You will have to decide how to distribute the gain between the two op amp stages in the circuit. It is best to put as much gain in early stages of a multi-stage amplifier as possible. However, when the gain of a single stage becomes very high, nonidealities of the op amp become bothersome. The first stage of the amplifier will behave reasonably well as long as the gain is less than about 10<sup>6</sup> &Omega;.<br />
<br />
# '''Analyze the photodiode amplifier circuit. Choose resistor values to achieve an overall gain that will yield an output of about 5 V when the input is 500 nA.'''<br />
# Construct the circuit on your breadboard.<br />
#* Disable the power supply before you build.<br />
#* Do not yet connect the photodiode.<br />
#* Orient the LF411 op-amps so that pin 1, which is marked with a dot on the package, is at the upper left.<br />
# Connect the +/- 15 V bus strips to power the op-amps power pins.<br />
# Connect the vertical blue bus strips to the horizontal blue bus strip.<br />
<br />
=====Test connectivity and basic operation=====<br />
<br />
# Verify that all your component connections are correct and match the design.<br />
# Check that wires and components are properly seated in the breadboard and not suspiciously loose.<br />
#* You can use the ohm-meter function of the digital multi-meter to verify continuity.<br />
# Turn on the power supply and verify that +15V appears on pin 7 and -15V on pin 4.<br />
# '''Short pins 2 and 3 (the V- and V+ inputs to the op-amp) with a small wire and measure the output voltages of IC1 and IC2.'''<br />
#* With the photodiode shorted the current signal is zero, but you may still see a significant offset voltage from 50 to 100 mV at IC1 pin 6.<br />
#* Refer to [http://measure.mit.edu/~20.309/wiki/index.php?title=Real_electronics#Real_op-amps Real Electronics, real op-amps] or ask an instructor to understand and address this non-ideal behavior.<br />
<br />
==Data acquisition hardware subsystem==<br />
<br />
{{:20.309:DAQ System}}<br />
<br />
====Summary of DAQ inputs/outputs====<br />
<br />
A special connector has been prepared for you, connected as shown below. The connections and cable wire colors are also summarized [http://dl.dropbox.com/u/12957607/DAQ_Cable_Assembly_for_DNA_melting_lab.pdf in a memo].<br />
<br />
{| class="wikitable"<br />
|-<br />
! Signal Name<br />
! Signal Location<br />
! Ground Location<br />
! Pin wire color<br />
|-<br />
! DAQ Inputs <br />
|-<br />
! RTD<br />
| AI0+<br />
| AI0-<br />
| +Orange / -Black [lone pair, not with third (red) wire]<br />
|-<br />
! Photodiode <br />
| AI1+ <br />
| AI1-<br />
| +Green / -White<br />
|}<br />
<br />
<center>[[Image:DAQ_Wire_Colors.png|450 px|thumb|center|DAQ Connection Cable]]</center><br />
<br />
==DNAMelter software==<br />
<br />
[[Image:BasicDNAMelterIcon.png|75 px|left]]<br />
Use the <code>Basic DNA Melter GUI</code> program (located on the lab computer desktop) to collect data from the apparatus.<br />
<br />
At the end of an experimental run, use the "Save" button to write the date to a file. The data will be tab-delimited and can be read into Matlab with the <code>load</code> command.<br />
<br />
Documentation for the DNAMelter software is available here: [[DNA Melting: Using the Basic DNAMelter GUI]]<br />
<br />
====If you need to debug the DAQ (skip otherwise!)====<br />
* When debugging problems, it is frequently useful to ascertain whether the data acquisition system is working properly. National Instruments provides an application for this purpose called Measurement and Automation Explorer. Use Measurement and Automation Explorer to verify that the DAQ is properly connected to and communicating with the computer. The program also provides the ability to manually control all of the DAQ system's inputs and outputs.<br />
** Launch Measurement and Automation Explorer by selecting Start->All Applications->National Instruments->Measurement and Automation.<br />
** In the "Configuration" tab, select "Devices and Interfaces" and then "NI-DAQMx Devices." Select "Dev1."<br />
** If Dev 1 does not show up, the problem is that computer cannot communicate with the DAQ system. Ensure that the USB cable is connected to the DAQ system. If the cable is connected and the device still does not show up, unplug the USB cable, count to five, and plug it back in. <br />
** If the DAQ shows up under a different name, disconnect the USB cable. Delete all the "NI USB-6xxx" entries and then reconnect the USB cable. Verify that one entry reappears and that it is called "Dev1." <br />
** Select the "Test Panels" tab to manually control or read signals from the DAQ.<br />
* If you run DNAMelter and there is a data acquisition error, for example, "<code>Data acquisition cannot start!</code>", open Measurement and Automation Explorer and follow the steps above. Run DNAMelter again. If it still doesn't work, grab a beer or ask an instructor for help.<br />
* The DAQ has only one analog-to-digital converter (ADC). A multiplexer circuit sequentially samples voltages from the analog inputs configured in the control software. If any of the inputs is outside the allowable range of +/-10V, all of the inputs may behave erratically. The voltages reported may look reasonable so this can be very misleading. If none of the DAQ readings make sense, ensure that all of the analog voltages are in the allowable range and reset the DAQ by cycling the power of the DAQ (use the switch on the 6341 or disconnect the USB of the 6212).<br />
* If the DAQ gets short-circuited, it may crash. If this happens, Matlab will frequently give a deceptive error message, for example claiming that the limits on the band-pass filter do not satisfy some condition. In this case, correct the connection and cycle the DAQ power.<br />
<br />
==Part 1 measurements==<br />
<br />
Please read this entire section before beginning. Then start at Testing the instrument, with reference back to the sample sections as-needed.<br />
<br />
===Making samples===<br />
<br />
{{Template:Biohazard warning|message=LC Green in DMSO is readily absorbed through skin. Synthetic oligonucleotides may be harmful by inhalation, ingestion, or skin absorption. Wear gloves when handling samples. Wear safety goggles at all times when pipetting the LC Green/DNA samples. Do not create aerosols. The health effects of LC Green have not been thoroughly investigated. See the LC Green and synthetic oligonucleotide under <code>../EHS Guidelines/MSDS Repository</code> in the course locker for more information.}}<br />
<br />
====Sample prep steps====<br />
<br />
#Pipet 500 &mu;L of DNA plus dye solution into a glass vial. <br />
#Pipet up to 200 &mu;L of mineral oil on top of the sample to help prevent evaporation. It will help to pipette into the corners first. Be careful not to get oil on the cuvette sides far above the sample. It will run down during your experiment and cause shifts in the photodiode signal. Place the cap loosely on the vial. <br />
#*Keep the sample vertical to make sure the oil stays on top. <br />
<br />
* You can use the same sample for several heating/cooling cycles.<br />
* Only discard a sample if you lose significant volume due to evaporation or if your signal gets too low. <br />
<br />
To clean your glass vial between samples, flush with alcohol in the waste container and rinse with water at the drain. Suck out residual liquid with the vacuum and drawn Pasteur pipette to the left of the sink.<br />
<br />
====Sample disposal====<br />
<br />
{{Template:Environmental Warning|message=Discard pipette tips with DNA sample residue in the pipette tips or the ''Biohazard Sharps'' container. Do not pour synthetic oligonucleotides with LC Green down the drain. Pour your used samples into the waste container provided in the middle of the wet bench.}}<br />
<br />
===Testing the instrument===<br />
<br />
Once your apparatus is built, use samples of fluorescein (~ 30&mu;M concentration) as a stand-in for LC Green + DNA to test the instrument. Fluorescein is cheap, non-toxic and will not bleach as quickly as LC Green.<br />
<br />
====Making a simple measurement of your instrument's low frequency SNR====<br />
<br />
Carry out a simple estimate of the signal to noise ratio (SNR) of the instrument by comparing the difference between the average signal with a sample present and with water as a null reference. This process requires a vial containing 500 uL of 30 uM fluorescein and a vial containing the same amount of water. <br />
<br />
# Run the <code>Basic DNA Melter GUI</code>. <br />
# Place a vial of DI water in your instrument.<br />
# Clear the data and wait 10 seconds.<br />
# Replace the water vial with the DNA of fluorescein vial, record for another 10 seconds, then save the data. <br />
#* Be sure that all other conditions, such as temperature, are stable throughout the test. <br />
# Segment your data into that when the water was present and that when the fluorescein was present. Calculate the average signal in each case. Record the difference between the two averages as your signal. <br />
# Now discard the water portion of your signal and subtract the average fluorescein signal value from the fluorescein signal.<br />
# Measure the standard deviation of this residual and record as record as your estimate of the noise level.<br />
# '''Calculate the SNR as signal divided by noise and record in your lab report.'''<br />
<br />
===Experiment steps===<br />
<br />
Once the instrument is confirmed to be functioning properly, follow these instructions to generate melting curve data for the 20 bp DNA-LC Green solution.<br />
<br />
#Open and run the <code>Basic DNA Melter GUI</code> in Matlab and follow the instructions there.<br />
# Confirm that block temperature is near room temperature when the Diablotek supply is off.<br />
# Apply heat to the block by switching the Diablotek on to increase the block temperature to 95&deg;C, toggle the Diablotek power in order to hold the temperature long enough for the fluorescence curve to reach a minimum, then allow the block to cool down by switching off the Diablotek.<br />
#* Other approaches may be used. One goal would be to heat the block more slowly, possibly by toggling the current while heating.<br />
# Now prepare a sample using the stock of "beater" DNA.<br />
# Place the vial in the instrument, but keep the LED off until ready to measure the melting curve.<br />
# Measure melting curves of the beater DNA sample as-desired to understand instrument operation.<br />
# Prepare a sample using fresh 20 bp 100 mM KCl DNA.<br />
# Measure a melting curve of the fresh sample.<br />
#* Be sure to save a copy in a local directory.<br />
<br />
{{:DNA Melting Report Requirements for Part 1}}<br />
<br />
==Lab manual sections==<br />
<br />
*[[Lab Manual:Measuring DNA Melting Curves]]<br />
*[[DNA Melting: Simulating DNA Melting - Basics]]<br />
*[[DNA Melting Part 1: Measuring Temperature and Fluorescence]]<br />
*[[DNA Melting Report Requirements for Part 1]]<br />
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]<br />
*[[DNA Melting Report Requirements for Part 2]]<br />
<br />
==References==<br />
<references/><br />
<br />
==Subset of datasheets==<br />
(Many more can be found online or on the course share)<br />
#[http://www.ni.com/pdf/manuals/371931f.pdf National Instruments USB-6212 user manual]<br />
#[http://www.ni.com/pdf/manuals/370784d.pdf National Instruments USB-6341 user manual]<br />
#[http://www.national.com/ds/LF/LF411.pdf Op-amp datasheet]<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/DNA_Melting_Report_Requirements_for_Part_1DNA Melting Report Requirements for Part 12015-11-04T21:29:07Z<p>Steven Nagle: </p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:DNA Melting Lab]]<br />
<br />
==Report requirements==<br />
* One member of your group should submit a single PDF file to Stellar in advance of the deadline. The filename should consist of the last names of all group members, CamelCased, in alphabetical order, with a .pdf extension. Example: <code>CrickFranklinWatson.pdf</code>.<br />
* The file must be less than 20 MB.<br />
* Include code at the end of the document in an appendix, in the same pdf file, not as as separate upload.<br />
<br />
==Part 1 report outline==<br />
# Document your circuit design, optical design, and any ways that your instrument differs from the system described in the lab manual.<br />
#* For example, record your choice of resistors, capacitors and lenses, as well as a diagram of your chosen optical components.<br />
#* If you have not modified the circuits from their form in the report, you do not need to include the schematic in your report.<br />
#* Include a picture of your instrument.<br />
# Report your signal to noise measurement.<br />
# Plot at least one melting curve. <br />
#* The plot should have temperature in &deg;C on the horizontal axis and fraction of double stranded DNA on the vertical axis.<br />
#* On the same set of axes, include a simulated curve generated by DINAMelt, OligoCalc, or another software simulator.<br />
#* Also on the same set of axes, plot the output of the <tt>DnaFraction</tt> function evaluated with best-fit parameters. You may use nlinfit to choose the best-fit parameters or you may choose them manually.<br />
#* Include a legend.<br />
# Report the estimated melting temperature and the best-fit values of ''&Delta;H&deg;'', ''&Delta;S&deg;''.<br />
# Explain the statistical method you will use to identify your group's unknown sample in part 2 of this lab.<br />
#* State the acceptance/rejection criteria for any hypotheses tests you will use.<br />
<br />
==Lab manual sections==<br />
<br />
*[[Lab Manual:Measuring DNA Melting Curves]]<br />
*[[DNA Melting: Simulating DNA Melting - Basics]]<br />
*[[DNA Melting Part 1: Measuring Temperature and Fluorescence]]<br />
*[[DNA Melting Report Requirements for Part 1]]<br />
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]<br />
*[[DNA Melting Report Requirements for Part 2]]<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/DNA_Melting_Part_1:_Measuring_Temperature_and_FluorescenceDNA Melting Part 1: Measuring Temperature and Fluorescence2015-11-04T21:16:34Z<p>Steven Nagle: /* Temperature detection circuit */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
<br />
[[Image:DNA Melting Block Diagram.jpg|frame|DNA melting apparatus block diagram showing the five functional groups of the instrument.]]<br />
<br />
<br />
''In theory, there is no difference between theory and practice. But, in practice, there is.'' <br />
<br />
''&mdash;[http://en.wikiquote.org/wiki/Jan_L._A._van_de_Snepscheut Jan L. A. van de Snepscheut]/[http://en.wikiquote.org/wiki/Yogi_Berra Yogi Berra]/and or [http://c2.com/cgi/wiki?DifferenceBetweenTheoryAndPractice Chuck Reid]'' <br />
<br />
You will construct a basic DNA melting instrument consisting of the five functional groups shown in the block diagram:<br />
* excitation<br />
* heating<br />
* fluorescent detection<br />
* temperature measurement<br />
* data acquisition and control<br />
<br />
A metal block will hold a sample vial in the excitation light path and transfer heat to the sample. The sample contains a combination of DNA, SYBR Green I and a salt. Recall that SYBR Green I fluoresces when it is bound to double stranded DNA (dsDNA) by a factor of up to 1000x compared to when it is bound to single stranded DNA (ssDNA). Detection of this fluorescence provides a relative measure of the fraction of dsDNA to total DNA, which can be compared to predictions from a thermodynamic model.<br />
<br />
The dye has a peak sensitivity to blue light at 497 nm so a blue LED will be used to illuminate the sample. The dye emits green light with an emission peak at 520 nm. A photodiode will be placed at 90&deg; to the LED source to detect the green light emitted by SYBR Green I. Because LEDs have a fairly broad spectrum, first a moderately narrow band-pass filter will be used to limit the excitation wavelength to between 450 and 490 nm. Then a complimentary long-pass emission filter will be placed between the sample and detector to pass only wavelengths longer than 515 nm. This filter combination, when used with this dye system, eliminates any background signal due to the excitation light.<br />
<br />
The sample block will be heated by a TEC (thermoelectric cooler) and its temperature measured by an embedded RTD (resistance temperature detector). Also, since photodiodes produce a very small amount of current, a high gain amplifier will convert the photodiode current into a measurable voltage. The DAQ (data acquisition card) and PC will digitize the amplified photodiode and RTD signals. A Matlab program is provided to record these fluorescence and temperature signals over time and save the data to a file. The file can then be loaded into Matlab for analysis, or if you prefer, Python or equivalent where the signals will be converted into dsDNA concentration and sample temperature. Finally, the dsDNA sample melting temperature will be estimated from a plot of df/dT vs T (''f'' represents the fraction of total DNA that is in double-stranded form) and from a comparison to the thermodynamic model.<br />
<br />
The functional groups in the block diagram are one of two fundamental frameworks used to describe an instrument. The other sub-divides the instrument into categories that often mirror historical areas of engineering. The former is focused on what will be done, including the overall scientific goals and the physical processes to be measured. The latter is focused on how it will be done, e.g. using optics, electronics or perhaps a practical knowledge of thermal management. A good engineer will constantly be thinking of the instrument in both frameworks. <br />
<br />
==Mechanical subsystem==<br />
<br />
[[Image:DNA_Setup_v2_Iso.png|600 px|thumb|center|DNA Melting System Model]]<br />
<br />
Mechanical support for the heating block, sample and optics will be built on a portable optical breadboard from [http://www.thorlabs.com ThorLabs]. Most of the components in the lab are from this supplier because the selection is large, the quality is quite good and the price reasonable. Although you are free to modify the design as the lab progresses, begin a build from the standard design shown above and demonstrated by the example in the lab. Gather the following Thorlabs components but do not start to assemble.<br />
<br />
* 1 MB1224 optical breadboard<br />
* 2 CL5 clamps (or similar)<br />
* 2 1/4-20 5/8" screws<br />
* 1 sheet metal support bracket<br />
* 4 1/4-20 1/4" screws<br />
<br />
==Thermal subsystem==<br />
The thermal subsystem includes all components to control heat flow into and out of the sample and monitor its temperature. Heating is accomplished using thermal electric coolers (TECs) in reverse. The hot side is placed in direct contact with the heating block, which is in direct contact with the sample vial. The TECs will be powered by manually controlling the Diablotek power supply. To avoid contamination of the sample or perturbation of its temperature, the temperature of a resistive temperature detector (RTD) embedded in the heating block will be monitored as a proxy for the sample temperature. In this part of the lab, air convection will cool down the sample when the Diablotek is switched off. The heat sink will help and the fan may be connected to a triple-output power supply to help, but these are included primarily for use in Part 2.<br />
<br />
===Parts===<br />
Gather the following components from the bins on the Center Cabinets, the DNA Melting drawer at the wet bench or on the East Cabinets:<br />
<br />
[[Image:CenterCabinets.jpg|right|250px|thumb|'''Center Cabinets''' The bins for most parts that you need will be set out on top of these cabinets, roughly in the center of the lab.]]<br />
[[Image:WetBench.jpg|right|250px|thumb|'''Wet Bench''' Supplies for our wet bench can be found in the three drawers at the left of the bench, the drawer left of the sink and on the wall behind and to the left as you face the wet bench. The drawer at the right of the wet bench contains various supplies for the DNA Melting instrument.]]<br />
[[Image:EastCabinets.jpg|right|250px|thumb|'''East Cabinets''' The center shelves of the East Cabinets contain the electronic breadboards, breadboard jumper wire, TEC connectors and TEC power supplies. Often these supplies are laid out on top of these cabinets.]]<br />
<br />
* A metal support bracket, having a fan, heatsink, 2 TEC heaters/coolers, and a heating block attached to it (if they're not attached, you'll need to attach them).<br />
* 3 wire nuts<br />
* 1 Diablotek 250 W power supply<br />
* 1 red/black TEC extension wire assembly<br />
<br />
===Assembly instructions===<br />
<br />
[[Image:TEC_picture.jpg|150 px|thumb|right|TEC showing hot side up when current flows into the red lead]]<br />
<br />
Ensure that the following have been done/attached to your metal bracket:<br />
# Attach the fan on the side of the support bracket using the 6-32 screws and nuts.<br />
# Attach the 2x2 heat sink to the bottom of the support bracket using two 4-40 screws at opposite corners.<br />
# Attach the support bracket to the MB1224 breadboard using 1/4-20 1/4" screws.<br />
# Now place the TECs on a napkin or two, with the red lead extending down and to the ''left'' as shown in the figure.<br />
# Apply a very thin layer of thermal grease to the exposed side of one of the TECs using a wooden stick from the drawer.<br />
#* Use a small amount of grease. Too much is worse than none.<br />
#* Best practice is to apply a baby-pea-sized amount, spread with the stick, smooth and remove excess almost to the point of total removal.<br />
# Now place the second TEC on top of the first.<br />
#* If the two TECs slide freely against each other, then there is too much grease.<br />
#** Separate the TECs and smooth/remove the grease until a modest force is required to slide them.<br />
#* Verify that the colors of the leads match. If not, flip one TEC.<br />
# Connect the black lead of the top TEC to the red lead of the bottom TEC using a wire nut.<br />
# Connect the TEC to the extension wire assembly using a wire nut to join the white wire to the red lead of the top TEC and the black wire to the black lead on the bottom TEC.<br />
# Join the white connector on the assembly to the black 2x2 connector of the Diabloteck.<br />
# Test the TECs by holding the them between thumb and finger while briefly flipping the switch on the Diablotek.<br />
#* One side should immediately heat up while the other immediately cools.<br />
#* The top side should be the hot side when the red wires extend down and to the '''left'''.<br />
#* If the Diablotek immediately shuts off (the fan will stop spinning) then there is a short. Check all connections.<br />
# Once the TEC assembly checks out, butter the bottom side of the assembly and place that side on the heat sink with the leads facing to the right.<br />
# Finally, apply thermal grease to the top side of the TEC assembly, place the heating block on top and anchor to the heat sink using the 1/2" long 4-40 screws and plastic washers.<br />
<br />
==Optical subsystem==<br />
<br />
A good optical subsystem design will be compact, stable and simple. It includes the optical components of the excitation and fluorescence detection functional groups from the block diagram. A good design will efficiently illuminate the sample and collect the maximum possible amount of emitted fluorescent light. There are a variety of ways to construct the apparatus. Use the model above and the example system in the lab as guides.<br />
<br />
The following is a rough set of instructions if you're not clear on how to replicate the example system in the lab. In addition to the following parts, you might want to add a few lenses to collimate your LED onto your sample, and to focus the light coming from your sample onto your photodiode. <br />
<br />
* 2 PH2 post holders<br />
* 2 BA1 post holder bases<br />
* 2 1/4-20 1/4" screws<br />
* 2 1/4-20 5/8" screws<br />
* 2 1/4" washers<br />
* 4 CP02 cage plates<br />
* 2 8-32 1/2" setscrews<br />
* 2 TR2 posts<br />
* 6 ER3 cage rails<br />
* 12 4-40 1/4" setscrews (or similar length)<br />
* 4 SML05 lens tubes<br />
* 4 SM1LTRR retaining rings<br />
* 1 blue LED PCB<br />
* 1 D470/40 excitation filter<br />
* 1 E515LPv2 emission filter<br />
* 1 SM1A6 adapter between SM1 and SM05 threads<br />
* 1 SM05PD1A mounted photodiode<br />
<br />
===Assembly instructions===<br />
<br />
# Attach each PH2 to a BA1 using 1/4" screws.<br />
# Attach two of the CP02 cage plates to two TR2 posts using an 8-32 setscrew.<br />
# Insert the TR2/CP02 assembly into the PH2/BA1 assemblies and lock in place with the TS25H setscrews.<br />
# Insert three ER4 cage rails into each CP02 above and lock in place with SS4S025 set screws or similar.<br />
#* Leave the blank hole on the bottom of the assembly.<br />
# Attach one of the remaining CP02 cage plates on the free end of each assembly above and lock in place.<br />
# Attach each of the above assemblies to the MB1224 using a washer and 1/4-20 screw less than 1" in length.<br />
# Arrange on the MB1224 and adjust CP02 heights as desired.<br />
# Mount the LED PCB in an SML05 lens tube and fix with a retaining ring.<br />
# Mount the excitation and emission filters in each of two lens tubes and fix with retaining ring.<br />
#* The arrow label on the thickness of the filter points ''toward the light source''.<br />
# Thread the SM1A6 adapter into a lens tube and fix with a retaining ring.<br />
# Thread the photodiode into the SM1A6 adapter.<br />
#* Teflon pipe thread tape can be used to hold the photodiode in the desired position.<br />
# Mount each lens tube in the appropriate CP02 cage plates.<br />
# Arrange each the resultant assemblies on the breadboard to most fully illuminate the sample vial and to gather the most of the resultant light on the photodiode.<br />
<br />
==Electronics subsystem==<br />
<br />
The electrical subsystem includes the LED with a current-limiting resistor, the photodiode, the transimpedance amplifier and a temperature detection circuit. The remainder of this section provides an example of each of these systems and instructions to build and test each one. Nevertheless, alternate ideas are welcome. Feel free to discuss with the Instructors.<br />
<br />
===Parts===<br />
<br />
Gather the following components from the electronics drawer to the right of the optics drawers, from the counter top opposite the wet bench and from the cable organizer on the East wall to the right of the white board.<br />
* 1 electronic breadboard<br />
* 1 jump wire kit<br />
* 2 red 3' banana cable or similar<br />
* 1 black 3' banana cable or similar<br />
* individual wire, as-needed from the scrap wire box or the wire spools opposite the wet bench<br />
* 1 62 &Omega; resistor from the center counter top on the South wall<br />
* 1 15 k&Omega; resistor<br />
* other resistors, as-needed for the amplifier<br />
* 1 blue LED from the DNA Melting drawer at the far right of the wet bench<br />
* 2 LF411 op-amps from the electronics drawer<br />
* 4 capacitors between 0.1 and 22 uF from either the electronics drawer or the counter top where the screws are stored.<br />
<br />
===Assembly instructions===<br />
<br />
====LED circuit====<br />
The led circuit consists of a 62 &Omega; resistor in series with a blue, jumbo LED. The circuit will be powered by the 5 volt fixed output of the lab power supply. The 62 &Omega; resistor prevents excessive current from flowing through the LED. Excessive current will let the smoke out of the LED.<br />
<br />
# Build the LED circuit on your electronic breadboard.<br />
#* Don't forget to connect the resistor. Directly connecting the LED to 5 V will blow out the LED.<br />
# Disable the output of the lab power supply.<br />
# Connect the LED circuit to the fixed 5 V output.<br />
<br />
====Temperature detection circuit====<br />
A platinum RTD is embedded inside the heating block. The resistance of the RTD varies with temperature according to the equation ''R<sub>RTD</sub>''=1000 &Omega; + 3.85 ''&theta;'', where ''&theta;'' is the temperature of the RTD in degrees Celsius. The easiest way to make a temperature measurement is to connect the RTD in series with a fixed resistor and a power supply. The output voltage of the resulting divider circuit varies as a function of temperature. <br />
<br />
The maximum current of the RTD is 1 mA. You will use a 15 V supply to the circuit, so the resistor must be at least 14 k&Omega;. Use a 15 k&Omega; resistor.<br />
<br />
# '''Find an equation for ''V<sub>out</sub>'' as a function of temperature. What voltage do you expect given an input voltage of 15 V with the RTD at room temperature.'''<br />
# Turn off the output of the triple-output power supply.<br />
# Connect the RTD to the electronic breadboard.<br />
# '''Measure the resistance of the 15 k&Omega; resistor with a DMM and record the value in your lab report.'''<br />
# Connect the 15k&Omega; resistor in series with the RTD.<br />
# Set the power supply for '''SERIES''' operation.<br />
# Set the output voltage to 15 V on the supply and make note of its exact value using the DMM.<br />
# Set the current limit to 0.1 A.<br />
# Connect the positive terminal of '''CH1''' to a banana terminal on the breadboard for +15 V and wire it to the top red bus strip for +15 V supply.<br />
# Connect the negative terminal of '''CH2''' to a banana terminal on the breadboard for -15 V.<br />
# Connect the positive terminal of '''CH2''' to the black terminal on the breadboard for a ground reference and wire it to the top blue bus strip.<br />
#* Use three jump wires to minimize the parasitic voltage drop due to the breadboard tie points.<br />
# Connect the +15 V bus strip to the second vertical, red bus strip and wire this bus strip to the top of your 15 k&Omega; resistor.<br />
# Turn on the output of the power supply and measure the voltage across the RTD using the DMM.<br />
# '''What is the measured voltage of the RTD at room temperature and how does it compare to your predictions above?'''<br />
# '''What is the temperature, based on your measured voltage and how does it compare with the clock on the wall (or some better measure)?'''<br />
# '''How is the temperature calculation affected by a 1% change in the 15 V input voltage?'''<br />
<br />
====Photodiode amplifier circuit====<br />
<br />
The current produced by the photodiode detector is a few tens of nanoamperes. This very small current must be amplified to a level where it can be easily measured. The data acquisition system you will use can measure voltages between + and -10 V. Use the two-stage op amp circuit shown below to convert the photodiode current to a voltage.<br />
<br />
<center>[[Image:Part1_TransimpedanceAmplifier.jpg|700 px|thumb|center|Multi-stage photodiode amplification circuit]]</center><br />
<br />
You will have to decide how to distribute the gain between the two op amp stages in the circuit. It is best to put as much gain in early stages of a multi-stage amplifier as possible. However, when the gain of a single stage becomes very high, nonidealities of the op amp become bothersome. The first stage of the amplifier will behave reasonably well as long as the gain is less than about 10<sup>6</sup> &Omega;.<br />
<br />
# '''Analyze the photodiode amplifier circuit. Choose resistor values to achieve an overall gain that will yield an output of about 5 V when the input is 500 nA.'''<br />
# Construct the circuit on your breadboard.<br />
#* Disable the power supply before you build.<br />
#* Do not yet connect the photodiode.<br />
#* Orient the LF411 op-amps so that pin 1, which is marked with a dot on the package, is at the upper left.<br />
# Connect the +/- 15 V bus strips to power the op-amps power pins.<br />
# Connect the vertical blue bus strips to the horizontal blue bus strip.<br />
<br />
=====Test connectivity and basic operation=====<br />
<br />
# Verify that all your component connections are correct and match the design.<br />
# Check that wires and components are properly seated in the breadboard and not suspiciously loose.<br />
#* You can use the ohm-meter function of the digital multi-meter to verify continuity.<br />
# Turn on the power supply and verify that +15V appears on pin 7 and -15V on pin 4.<br />
# '''Short pins 2 and 3 (the V- and V+ inputs to the op-amp) with a small wire and measure the output voltages of IC1 and IC2.'''<br />
#* With the photodiode shorted the current signal is zero, but you may still see a significant offset voltage from 50 to 100 mV at IC1 pin 6.<br />
#* Refer to [http://measure.mit.edu/~20.309/wiki/index.php?title=Real_electronics#Real_op-amps Real Electronics, real op-amps] or ask an instructor to understand and address this non-ideal behavior.<br />
<br />
==Data acquisition hardware subsystem==<br />
<br />
{{:20.309:DAQ System}}<br />
<br />
====Summary of DAQ inputs/outputs====<br />
<br />
A special connector has been prepared for you, connected as shown below. The connections and cable wire colors are also summarized [http://dl.dropbox.com/u/12957607/DAQ_Cable_Assembly_for_DNA_melting_lab.pdf in a memo].<br />
<br />
{| class="wikitable"<br />
|-<br />
! Signal Name<br />
! Signal Location<br />
! Ground Location<br />
! Pin wire color<br />
|-<br />
! DAQ Inputs <br />
|-<br />
! RTD<br />
| AI0+<br />
| AI0-<br />
| +Orange / -Black [lone pair, not with third (red) wire]<br />
|-<br />
! Photodiode <br />
| AI1+ <br />
| AI1-<br />
| +Green / -White<br />
|}<br />
<br />
<center>[[Image:DAQ_Wire_Colors.png|450 px|thumb|center|DAQ Connection Cable]]</center><br />
<br />
==DNAMelter software==<br />
<br />
[[Image:BasicDNAMelterIcon.png|75 px|left]]<br />
Use the <code>Basic DNA Melter GUI</code> program (located on the lab computer desktop) to collect data from the apparatus.<br />
<br />
At the end of an experimental run, use the "Save" button to write the date to a file. The data will be tab-delimited and can be read into Matlab with the <code>load</code> command.<br />
<br />
Documentation for the DNAMelter software is available here: [[DNA Melting: Using the Basic DNAMelter GUI]]<br />
<br />
====If you need to debug the DAQ (skip otherwise!)====<br />
* When debugging problems, it is frequently useful to ascertain whether the data acquisition system is working properly. National Instruments provides an application for this purpose called Measurement and Automation Explorer. Use Measurement and Automation Explorer to verify that the DAQ is properly connected to and communicating with the computer. The program also provides the ability to manually control all of the DAQ system's inputs and outputs.<br />
** Launch Measurement and Automation Explorer by selecting Start->All Applications->National Instruments->Measurement and Automation.<br />
** In the "Configuration" tab, select "Devices and Interfaces" and then "NI-DAQMx Devices." Select "Dev1."<br />
** If Dev 1 does not show up, the problem is that computer cannot communicate with the DAQ system. Ensure that the USB cable is connected to the DAQ system. If the cable is connected and the device still does not show up, unplug the USB cable, count to five, and plug it back in. <br />
** If the DAQ shows up under a different name, disconnect the USB cable. Delete all the "NI USB-6xxx" entries and then reconnect the USB cable. Verify that one entry reappears and that it is called "Dev1." <br />
** Select the "Test Panels" tab to manually control or read signals from the DAQ.<br />
* If you run DNAMelter and there is a data acquisition error, for example, "<code>Data acquisition cannot start!</code>", open Measurement and Automation Explorer and follow the steps above. Run DNAMelter again. If it still doesn't work, grab a beer or ask an instructor for help.<br />
* The DAQ has only one analog-to-digital converter (ADC). A multiplexer circuit sequentially samples voltages from the analog inputs configured in the control software. If any of the inputs is outside the allowable range of +/-10V, all of the inputs may behave erratically. The voltages reported may look reasonable so this can be very misleading. If none of the DAQ readings make sense, ensure that all of the analog voltages are in the allowable range and reset the DAQ by cycling the power of the DAQ (use the switch on the 6341 or disconnect the USB of the 6212).<br />
* If the DAQ gets short-circuited, it may crash. If this happens, Matlab will frequently give a deceptive error message, for example claiming that the limits on the band-pass filter do not satisfy some condition. In this case, correct the connection and cycle the DAQ power.<br />
<br />
==Part 1 measurements==<br />
<br />
Please read this entire section before beginning. Then start at Testing the instrument, with reference back to the sample sections as-needed.<br />
<br />
===Making samples===<br />
<br />
{{Template:Biohazard warning|message=LC Green in DMSO is readily absorbed through skin. Synthetic oligonucleotides may be harmful by inhalation, ingestion, or skin absorption. Wear gloves when handling samples. Wear safety goggles at all times when pipetting the LC Green/DNA samples. Do not create aerosols. The health effects of LC Green have not been thoroughly investigated. See the LC Green and synthetic oligonucleotide under <code>../EHS Guidelines/MSDS Repository</code> in the course locker for more information.}}<br />
<br />
====Sample prep steps====<br />
<br />
#Pipet 500 &mu;L of DNA plus dye solution into a glass vial. <br />
#Pipet up to 200 &mu;L of mineral oil on top of the sample to help prevent evaporation. It will help to pipette into the corners first. Be careful not to get oil on the cuvette sides far above the sample. It will run down during your experiment and cause shifts in the photodiode signal. Place the cap loosely on the vial. <br />
#*Keep the sample vertical to make sure the oil stays on top. <br />
<br />
* You can use the same sample for several heating/cooling cycles.<br />
* Only discard a sample if you lose significant volume due to evaporation or if your signal gets too low. <br />
<br />
To clean your glass vial between samples, flush with alcohol in the waste container and rinse with water at the drain. Suck out residual liquid with the vacuum and drawn Pasteur pipette to the left of the sink.<br />
<br />
====Sample disposal====<br />
<br />
{{Template:Environmental Warning|message=Discard pipette tips with DNA sample residue in the pipette tips or the ''Biohazard Sharps'' container. Do not pour synthetic oligonucleotides with LC Green down the drain. Pour your used samples into the waste container provided in the middle of the wet bench.}}<br />
<br />
===Testing the instrument===<br />
<br />
Once your apparatus is built, use samples of fluorescein (~ 30&mu;M concentration) as a stand-in for LC Green + DNA to test the instrument. Fluorescein is cheap, non-toxic and will not bleach as quickly as LC Green.<br />
<br />
====Making a simple measurement of your instrument's low frequency SNR====<br />
<br />
Carry out a simple estimate of the signal to noise ratio (SNR) of the instrument by comparing the difference between the average signal with a sample present and with water as a null reference. This process requires a vial containing 500 uL of 30 uM fluorescein and a vial containing the same amount of water. <br />
<br />
# Run the <code>Basic DNA Melter GUI</code>. <br />
# Place a vial of DI water in your instrument.<br />
# Clear the data and wait 10 seconds.<br />
# Replace the water vial with the DNA of fluorescein vial, record for another 10 seconds, then save the data. <br />
#* Be sure that all other conditions, such as temperature, are stable throughout the test. <br />
# Segment your data into that when the water was present and that when the fluorescein was present. Calculate the average signal in each case. Record the difference between the two averages as your signal. <br />
# Now discard the water portion of your signal and subtract the average fluorescein signal value from the fluorescein signal.<br />
# Measure the standard deviation of this residual and record as record as your estimate of the noise level.<br />
# '''Calculate the SNR as signal divided by noise and record in your lab report.'''<br />
<br />
===Experiment steps===<br />
<br />
Once the instrument is confirmed to be functioning properly, follow these instructions to generate melting curve data for the 20 bp DNA-LC Green solution.<br />
<br />
#Open and run the <code>Basic DNA Melter GUI</code> in Matlab and follow the instructions there.<br />
# Confirm that block temperature is near room temperature when the Diablotek supply is off.<br />
# Apply heat to the block by switching the Diablotek on to increase the block temperature to 95&deg;C, toggle the Diablotek power in order to hold the temperature long enough for the fluorescence curve to reach a minimum, then allow the block to cool down by switching off the Diablotek.<br />
#* Other approaches may be used. One goal would be to heat the block more slowly, possibly by toggling the current while heating.<br />
# Now prepare a sample using the stock of "beater" DNA.<br />
# Place the vial in the instrument, but keep the LED off until ready to measure the melting curve.<br />
# Measure melting curves of the beater DNA sample as-desired to understand instrument operation.<br />
# Prepare a sample using fresh 20 bp 100 mM KCl DNA.<br />
# Measure a melting curve of the fresh sample.<br />
#* Be sure to save a copy in a local directory.<br />
<br />
{{:DNA Melting Report Requirements for Part 1}}<br />
<br />
==Lab manual sections==<br />
<br />
*[[Lab Manual:Measuring DNA Melting Curves]]<br />
*[[DNA Melting: Simulating DNA Melting - Basics]]<br />
*[[DNA Melting Part 1: Measuring Temperature and Fluorescence]]<br />
*[[DNA Melting Report Requirements for Part 1]]<br />
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]<br />
*[[DNA Melting Report Requirements for Part 2]]<br />
<br />
==References==<br />
<references/><br />
<br />
==Subset of datasheets==<br />
(Many more can be found online or on the course share)<br />
#[http://www.ni.com/pdf/manuals/371931f.pdf National Instruments USB-6212 user manual]<br />
#[http://www.ni.com/pdf/manuals/370784d.pdf National Instruments USB-6341 user manual]<br />
#[http://www.national.com/ds/LF/LF411.pdf Op-amp datasheet]<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/DNA_Melting_Part_1:_Measuring_Temperature_and_FluorescenceDNA Melting Part 1: Measuring Temperature and Fluorescence2015-11-04T21:15:09Z<p>Steven Nagle: /* Making a simple measurement of your instrument's low frequency SNR */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
<br />
[[Image:DNA Melting Block Diagram.jpg|frame|DNA melting apparatus block diagram showing the five functional groups of the instrument.]]<br />
<br />
<br />
''In theory, there is no difference between theory and practice. But, in practice, there is.'' <br />
<br />
''&mdash;[http://en.wikiquote.org/wiki/Jan_L._A._van_de_Snepscheut Jan L. A. van de Snepscheut]/[http://en.wikiquote.org/wiki/Yogi_Berra Yogi Berra]/and or [http://c2.com/cgi/wiki?DifferenceBetweenTheoryAndPractice Chuck Reid]'' <br />
<br />
You will construct a basic DNA melting instrument consisting of the five functional groups shown in the block diagram:<br />
* excitation<br />
* heating<br />
* fluorescent detection<br />
* temperature measurement<br />
* data acquisition and control<br />
<br />
A metal block will hold a sample vial in the excitation light path and transfer heat to the sample. The sample contains a combination of DNA, SYBR Green I and a salt. Recall that SYBR Green I fluoresces when it is bound to double stranded DNA (dsDNA) by a factor of up to 1000x compared to when it is bound to single stranded DNA (ssDNA). Detection of this fluorescence provides a relative measure of the fraction of dsDNA to total DNA, which can be compared to predictions from a thermodynamic model.<br />
<br />
The dye has a peak sensitivity to blue light at 497 nm so a blue LED will be used to illuminate the sample. The dye emits green light with an emission peak at 520 nm. A photodiode will be placed at 90&deg; to the LED source to detect the green light emitted by SYBR Green I. Because LEDs have a fairly broad spectrum, first a moderately narrow band-pass filter will be used to limit the excitation wavelength to between 450 and 490 nm. Then a complimentary long-pass emission filter will be placed between the sample and detector to pass only wavelengths longer than 515 nm. This filter combination, when used with this dye system, eliminates any background signal due to the excitation light.<br />
<br />
The sample block will be heated by a TEC (thermoelectric cooler) and its temperature measured by an embedded RTD (resistance temperature detector). Also, since photodiodes produce a very small amount of current, a high gain amplifier will convert the photodiode current into a measurable voltage. The DAQ (data acquisition card) and PC will digitize the amplified photodiode and RTD signals. A Matlab program is provided to record these fluorescence and temperature signals over time and save the data to a file. The file can then be loaded into Matlab for analysis, or if you prefer, Python or equivalent where the signals will be converted into dsDNA concentration and sample temperature. Finally, the dsDNA sample melting temperature will be estimated from a plot of df/dT vs T (''f'' represents the fraction of total DNA that is in double-stranded form) and from a comparison to the thermodynamic model.<br />
<br />
The functional groups in the block diagram are one of two fundamental frameworks used to describe an instrument. The other sub-divides the instrument into categories that often mirror historical areas of engineering. The former is focused on what will be done, including the overall scientific goals and the physical processes to be measured. The latter is focused on how it will be done, e.g. using optics, electronics or perhaps a practical knowledge of thermal management. A good engineer will constantly be thinking of the instrument in both frameworks. <br />
<br />
==Mechanical subsystem==<br />
<br />
[[Image:DNA_Setup_v2_Iso.png|600 px|thumb|center|DNA Melting System Model]]<br />
<br />
Mechanical support for the heating block, sample and optics will be built on a portable optical breadboard from [http://www.thorlabs.com ThorLabs]. Most of the components in the lab are from this supplier because the selection is large, the quality is quite good and the price reasonable. Although you are free to modify the design as the lab progresses, begin a build from the standard design shown above and demonstrated by the example in the lab. Gather the following Thorlabs components but do not start to assemble.<br />
<br />
* 1 MB1224 optical breadboard<br />
* 2 CL5 clamps (or similar)<br />
* 2 1/4-20 5/8" screws<br />
* 1 sheet metal support bracket<br />
* 4 1/4-20 1/4" screws<br />
<br />
==Thermal subsystem==<br />
The thermal subsystem includes all components to control heat flow into and out of the sample and monitor its temperature. Heating is accomplished using thermal electric coolers (TECs) in reverse. The hot side is placed in direct contact with the heating block, which is in direct contact with the sample vial. The TECs will be powered by manually controlling the Diablotek power supply. To avoid contamination of the sample or perturbation of its temperature, the temperature of a resistive temperature detector (RTD) embedded in the heating block will be monitored as a proxy for the sample temperature. In this part of the lab, air convection will cool down the sample when the Diablotek is switched off. The heat sink will help and the fan may be connected to a triple-output power supply to help, but these are included primarily for use in Part 2.<br />
<br />
===Parts===<br />
Gather the following components from the bins on the Center Cabinets, the DNA Melting drawer at the wet bench or on the East Cabinets:<br />
<br />
[[Image:CenterCabinets.jpg|right|250px|thumb|'''Center Cabinets''' The bins for most parts that you need will be set out on top of these cabinets, roughly in the center of the lab.]]<br />
[[Image:WetBench.jpg|right|250px|thumb|'''Wet Bench''' Supplies for our wet bench can be found in the three drawers at the left of the bench, the drawer left of the sink and on the wall behind and to the left as you face the wet bench. The drawer at the right of the wet bench contains various supplies for the DNA Melting instrument.]]<br />
[[Image:EastCabinets.jpg|right|250px|thumb|'''East Cabinets''' The center shelves of the East Cabinets contain the electronic breadboards, breadboard jumper wire, TEC connectors and TEC power supplies. Often these supplies are laid out on top of these cabinets.]]<br />
<br />
* A metal support bracket, having a fan, heatsink, 2 TEC heaters/coolers, and a heating block attached to it (if they're not attached, you'll need to attach them).<br />
* 3 wire nuts<br />
* 1 Diablotek 250 W power supply<br />
* 1 red/black TEC extension wire assembly<br />
<br />
===Assembly instructions===<br />
<br />
[[Image:TEC_picture.jpg|150 px|thumb|right|TEC showing hot side up when current flows into the red lead]]<br />
<br />
Ensure that the following have been done/attached to your metal bracket:<br />
# Attach the fan on the side of the support bracket using the 6-32 screws and nuts.<br />
# Attach the 2x2 heat sink to the bottom of the support bracket using two 4-40 screws at opposite corners.<br />
# Attach the support bracket to the MB1224 breadboard using 1/4-20 1/4" screws.<br />
# Now place the TECs on a napkin or two, with the red lead extending down and to the ''left'' as shown in the figure.<br />
# Apply a very thin layer of thermal grease to the exposed side of one of the TECs using a wooden stick from the drawer.<br />
#* Use a small amount of grease. Too much is worse than none.<br />
#* Best practice is to apply a baby-pea-sized amount, spread with the stick, smooth and remove excess almost to the point of total removal.<br />
# Now place the second TEC on top of the first.<br />
#* If the two TECs slide freely against each other, then there is too much grease.<br />
#** Separate the TECs and smooth/remove the grease until a modest force is required to slide them.<br />
#* Verify that the colors of the leads match. If not, flip one TEC.<br />
# Connect the black lead of the top TEC to the red lead of the bottom TEC using a wire nut.<br />
# Connect the TEC to the extension wire assembly using a wire nut to join the white wire to the red lead of the top TEC and the black wire to the black lead on the bottom TEC.<br />
# Join the white connector on the assembly to the black 2x2 connector of the Diabloteck.<br />
# Test the TECs by holding the them between thumb and finger while briefly flipping the switch on the Diablotek.<br />
#* One side should immediately heat up while the other immediately cools.<br />
#* The top side should be the hot side when the red wires extend down and to the '''left'''.<br />
#* If the Diablotek immediately shuts off (the fan will stop spinning) then there is a short. Check all connections.<br />
# Once the TEC assembly checks out, butter the bottom side of the assembly and place that side on the heat sink with the leads facing to the right.<br />
# Finally, apply thermal grease to the top side of the TEC assembly, place the heating block on top and anchor to the heat sink using the 1/2" long 4-40 screws and plastic washers.<br />
<br />
==Optical subsystem==<br />
<br />
A good optical subsystem design will be compact, stable and simple. It includes the optical components of the excitation and fluorescence detection functional groups from the block diagram. A good design will efficiently illuminate the sample and collect the maximum possible amount of emitted fluorescent light. There are a variety of ways to construct the apparatus. Use the model above and the example system in the lab as guides.<br />
<br />
The following is a rough set of instructions if you're not clear on how to replicate the example system in the lab. In addition to the following parts, you might want to add a few lenses to collimate your LED onto your sample, and to focus the light coming from your sample onto your photodiode. <br />
<br />
* 2 PH2 post holders<br />
* 2 BA1 post holder bases<br />
* 2 1/4-20 1/4" screws<br />
* 2 1/4-20 5/8" screws<br />
* 2 1/4" washers<br />
* 4 CP02 cage plates<br />
* 2 8-32 1/2" setscrews<br />
* 2 TR2 posts<br />
* 6 ER3 cage rails<br />
* 12 4-40 1/4" setscrews (or similar length)<br />
* 4 SML05 lens tubes<br />
* 4 SM1LTRR retaining rings<br />
* 1 blue LED PCB<br />
* 1 D470/40 excitation filter<br />
* 1 E515LPv2 emission filter<br />
* 1 SM1A6 adapter between SM1 and SM05 threads<br />
* 1 SM05PD1A mounted photodiode<br />
<br />
===Assembly instructions===<br />
<br />
# Attach each PH2 to a BA1 using 1/4" screws.<br />
# Attach two of the CP02 cage plates to two TR2 posts using an 8-32 setscrew.<br />
# Insert the TR2/CP02 assembly into the PH2/BA1 assemblies and lock in place with the TS25H setscrews.<br />
# Insert three ER4 cage rails into each CP02 above and lock in place with SS4S025 set screws or similar.<br />
#* Leave the blank hole on the bottom of the assembly.<br />
# Attach one of the remaining CP02 cage plates on the free end of each assembly above and lock in place.<br />
# Attach each of the above assemblies to the MB1224 using a washer and 1/4-20 screw less than 1" in length.<br />
# Arrange on the MB1224 and adjust CP02 heights as desired.<br />
# Mount the LED PCB in an SML05 lens tube and fix with a retaining ring.<br />
# Mount the excitation and emission filters in each of two lens tubes and fix with retaining ring.<br />
#* The arrow label on the thickness of the filter points ''toward the light source''.<br />
# Thread the SM1A6 adapter into a lens tube and fix with a retaining ring.<br />
# Thread the photodiode into the SM1A6 adapter.<br />
#* Teflon pipe thread tape can be used to hold the photodiode in the desired position.<br />
# Mount each lens tube in the appropriate CP02 cage plates.<br />
# Arrange each the resultant assemblies on the breadboard to most fully illuminate the sample vial and to gather the most of the resultant light on the photodiode.<br />
<br />
==Electronics subsystem==<br />
<br />
The electrical subsystem includes the LED with a current-limiting resistor, the photodiode, the transimpedance amplifier and a temperature detection circuit. The remainder of this section provides an example of each of these systems and instructions to build and test each one. Nevertheless, alternate ideas are welcome. Feel free to discuss with the Instructors.<br />
<br />
===Parts===<br />
<br />
Gather the following components from the electronics drawer to the right of the optics drawers, from the counter top opposite the wet bench and from the cable organizer on the East wall to the right of the white board.<br />
* 1 electronic breadboard<br />
* 1 jump wire kit<br />
* 2 red 3' banana cable or similar<br />
* 1 black 3' banana cable or similar<br />
* individual wire, as-needed from the scrap wire box or the wire spools opposite the wet bench<br />
* 1 62 &Omega; resistor from the center counter top on the South wall<br />
* 1 15 k&Omega; resistor<br />
* other resistors, as-needed for the amplifier<br />
* 1 blue LED from the DNA Melting drawer at the far right of the wet bench<br />
* 2 LF411 op-amps from the electronics drawer<br />
* 4 capacitors between 0.1 and 22 uF from either the electronics drawer or the counter top where the screws are stored.<br />
<br />
===Assembly instructions===<br />
<br />
====LED circuit====<br />
The led circuit consists of a 62 &Omega; resistor in series with a blue, jumbo LED. The circuit will be powered by the 5 volt fixed output of the lab power supply. The 62 &Omega; resistor prevents excessive current from flowing through the LED. Excessive current will let the smoke out of the LED.<br />
<br />
# Build the LED circuit on your electronic breadboard.<br />
#* Don't forget to connect the resistor. Directly connecting the LED to 5 V will blow out the LED.<br />
# Disable the output of the lab power supply.<br />
# Connect the LED circuit to the fixed 5 V output.<br />
<br />
====Temperature detection circuit====<br />
A platinum RTD is embedded inside the heating block. The resistance of the RTD varies with temperature according to the equation ''R<sub>RTD</sub>''=1000 &Omega; + 3.85 ''&theta;'', where ''&theta;'' is the temperature of the RTD in degrees Celsius. The easiest way to make a temperature measurement is to connect the RTD in series with a fixed resistor and a power supply. The output voltage of the resulting divider circuit varies as a function of temperature. <br />
<br />
The maximum current of the RTD is 1 mA. You will use a 15 V supply to the circuit, so the resistor must be at least 14 k&Omega;. Use a 15 k&Omega; resistor.<br />
<br />
# '''Find an equation for ''V<sub>out</sub>'' as a function of temperature. What voltage do you expect given an input voltage of 15 V with the RTD at room temperature.'''<br />
# Turn off the output of the triple-output power supply.<br />
# Connect the RTD to the electronic breadboard.<br />
# '''Measure the resistance of the 15 k&Omega; resistor with a DMM and record the value.'''<br />
# Connect the 15k&Omega; resistor in series with the RTD.<br />
# Set the power supply for '''SERIES''' operation.<br />
# Set the output voltage to 15 V on the supply and make note of its exact value using the DMM.<br />
# Set the current limit to 0.1 A.<br />
# Connect the positive terminal of '''CH1''' to a banana terminal on the breadboard for +15 V and wire it to the top red bus strip for +15 V supply.<br />
# Connect the negative terminal of '''CH2''' to a banana terminal on the breadboard for -15 V.<br />
# Connect the positive terminal of '''CH2''' to the black terminal on the breadboard for a ground reference and wire it to the top blue bus strip.<br />
#* Use three jump wires to minimize the parasitic voltage drop due to the breadboard tie points.<br />
# Connect the +15 V bus strip to the second vertical, red bus strip and wire this bus strip to the top of your 15 k&Omega; resistor.<br />
# Turn on the output of the power supply and measure the voltage across the RTD using the DMM.<br />
# '''What is the measured voltage of the RTD at room temperature and how does it compare to your predictions above?'''<br />
# '''What is the temperature, based on your measured voltage and how does it compare with the clock on the wall (or some better measure)?'''<br />
# '''How is the temperature calculation affected by a 1% change in the 15 V input voltage?'''<br />
<br />
====Photodiode amplifier circuit====<br />
<br />
The current produced by the photodiode detector is a few tens of nanoamperes. This very small current must be amplified to a level where it can be easily measured. The data acquisition system you will use can measure voltages between + and -10 V. Use the two-stage op amp circuit shown below to convert the photodiode current to a voltage.<br />
<br />
<center>[[Image:Part1_TransimpedanceAmplifier.jpg|700 px|thumb|center|Multi-stage photodiode amplification circuit]]</center><br />
<br />
You will have to decide how to distribute the gain between the two op amp stages in the circuit. It is best to put as much gain in early stages of a multi-stage amplifier as possible. However, when the gain of a single stage becomes very high, nonidealities of the op amp become bothersome. The first stage of the amplifier will behave reasonably well as long as the gain is less than about 10<sup>6</sup> &Omega;.<br />
<br />
# '''Analyze the photodiode amplifier circuit. Choose resistor values to achieve an overall gain that will yield an output of about 5 V when the input is 500 nA.'''<br />
# Construct the circuit on your breadboard.<br />
#* Disable the power supply before you build.<br />
#* Do not yet connect the photodiode.<br />
#* Orient the LF411 op-amps so that pin 1, which is marked with a dot on the package, is at the upper left.<br />
# Connect the +/- 15 V bus strips to power the op-amps power pins.<br />
# Connect the vertical blue bus strips to the horizontal blue bus strip.<br />
<br />
=====Test connectivity and basic operation=====<br />
<br />
# Verify that all your component connections are correct and match the design.<br />
# Check that wires and components are properly seated in the breadboard and not suspiciously loose.<br />
#* You can use the ohm-meter function of the digital multi-meter to verify continuity.<br />
# Turn on the power supply and verify that +15V appears on pin 7 and -15V on pin 4.<br />
# '''Short pins 2 and 3 (the V- and V+ inputs to the op-amp) with a small wire and measure the output voltages of IC1 and IC2.'''<br />
#* With the photodiode shorted the current signal is zero, but you may still see a significant offset voltage from 50 to 100 mV at IC1 pin 6.<br />
#* Refer to [http://measure.mit.edu/~20.309/wiki/index.php?title=Real_electronics#Real_op-amps Real Electronics, real op-amps] or ask an instructor to understand and address this non-ideal behavior.<br />
<br />
==Data acquisition hardware subsystem==<br />
<br />
{{:20.309:DAQ System}}<br />
<br />
====Summary of DAQ inputs/outputs====<br />
<br />
A special connector has been prepared for you, connected as shown below. The connections and cable wire colors are also summarized [http://dl.dropbox.com/u/12957607/DAQ_Cable_Assembly_for_DNA_melting_lab.pdf in a memo].<br />
<br />
{| class="wikitable"<br />
|-<br />
! Signal Name<br />
! Signal Location<br />
! Ground Location<br />
! Pin wire color<br />
|-<br />
! DAQ Inputs <br />
|-<br />
! RTD<br />
| AI0+<br />
| AI0-<br />
| +Orange / -Black [lone pair, not with third (red) wire]<br />
|-<br />
! Photodiode <br />
| AI1+ <br />
| AI1-<br />
| +Green / -White<br />
|}<br />
<br />
<center>[[Image:DAQ_Wire_Colors.png|450 px|thumb|center|DAQ Connection Cable]]</center><br />
<br />
==DNAMelter software==<br />
<br />
[[Image:BasicDNAMelterIcon.png|75 px|left]]<br />
Use the <code>Basic DNA Melter GUI</code> program (located on the lab computer desktop) to collect data from the apparatus.<br />
<br />
At the end of an experimental run, use the "Save" button to write the date to a file. The data will be tab-delimited and can be read into Matlab with the <code>load</code> command.<br />
<br />
Documentation for the DNAMelter software is available here: [[DNA Melting: Using the Basic DNAMelter GUI]]<br />
<br />
====If you need to debug the DAQ (skip otherwise!)====<br />
* When debugging problems, it is frequently useful to ascertain whether the data acquisition system is working properly. National Instruments provides an application for this purpose called Measurement and Automation Explorer. Use Measurement and Automation Explorer to verify that the DAQ is properly connected to and communicating with the computer. The program also provides the ability to manually control all of the DAQ system's inputs and outputs.<br />
** Launch Measurement and Automation Explorer by selecting Start->All Applications->National Instruments->Measurement and Automation.<br />
** In the "Configuration" tab, select "Devices and Interfaces" and then "NI-DAQMx Devices." Select "Dev1."<br />
** If Dev 1 does not show up, the problem is that computer cannot communicate with the DAQ system. Ensure that the USB cable is connected to the DAQ system. If the cable is connected and the device still does not show up, unplug the USB cable, count to five, and plug it back in. <br />
** If the DAQ shows up under a different name, disconnect the USB cable. Delete all the "NI USB-6xxx" entries and then reconnect the USB cable. Verify that one entry reappears and that it is called "Dev1." <br />
** Select the "Test Panels" tab to manually control or read signals from the DAQ.<br />
* If you run DNAMelter and there is a data acquisition error, for example, "<code>Data acquisition cannot start!</code>", open Measurement and Automation Explorer and follow the steps above. Run DNAMelter again. If it still doesn't work, grab a beer or ask an instructor for help.<br />
* The DAQ has only one analog-to-digital converter (ADC). A multiplexer circuit sequentially samples voltages from the analog inputs configured in the control software. If any of the inputs is outside the allowable range of +/-10V, all of the inputs may behave erratically. The voltages reported may look reasonable so this can be very misleading. If none of the DAQ readings make sense, ensure that all of the analog voltages are in the allowable range and reset the DAQ by cycling the power of the DAQ (use the switch on the 6341 or disconnect the USB of the 6212).<br />
* If the DAQ gets short-circuited, it may crash. If this happens, Matlab will frequently give a deceptive error message, for example claiming that the limits on the band-pass filter do not satisfy some condition. In this case, correct the connection and cycle the DAQ power.<br />
<br />
==Part 1 measurements==<br />
<br />
Please read this entire section before beginning. Then start at Testing the instrument, with reference back to the sample sections as-needed.<br />
<br />
===Making samples===<br />
<br />
{{Template:Biohazard warning|message=LC Green in DMSO is readily absorbed through skin. Synthetic oligonucleotides may be harmful by inhalation, ingestion, or skin absorption. Wear gloves when handling samples. Wear safety goggles at all times when pipetting the LC Green/DNA samples. Do not create aerosols. The health effects of LC Green have not been thoroughly investigated. See the LC Green and synthetic oligonucleotide under <code>../EHS Guidelines/MSDS Repository</code> in the course locker for more information.}}<br />
<br />
====Sample prep steps====<br />
<br />
#Pipet 500 &mu;L of DNA plus dye solution into a glass vial. <br />
#Pipet up to 200 &mu;L of mineral oil on top of the sample to help prevent evaporation. It will help to pipette into the corners first. Be careful not to get oil on the cuvette sides far above the sample. It will run down during your experiment and cause shifts in the photodiode signal. Place the cap loosely on the vial. <br />
#*Keep the sample vertical to make sure the oil stays on top. <br />
<br />
* You can use the same sample for several heating/cooling cycles.<br />
* Only discard a sample if you lose significant volume due to evaporation or if your signal gets too low. <br />
<br />
To clean your glass vial between samples, flush with alcohol in the waste container and rinse with water at the drain. Suck out residual liquid with the vacuum and drawn Pasteur pipette to the left of the sink.<br />
<br />
====Sample disposal====<br />
<br />
{{Template:Environmental Warning|message=Discard pipette tips with DNA sample residue in the pipette tips or the ''Biohazard Sharps'' container. Do not pour synthetic oligonucleotides with LC Green down the drain. Pour your used samples into the waste container provided in the middle of the wet bench.}}<br />
<br />
===Testing the instrument===<br />
<br />
Once your apparatus is built, use samples of fluorescein (~ 30&mu;M concentration) as a stand-in for LC Green + DNA to test the instrument. Fluorescein is cheap, non-toxic and will not bleach as quickly as LC Green.<br />
<br />
====Making a simple measurement of your instrument's low frequency SNR====<br />
<br />
Carry out a simple estimate of the signal to noise ratio (SNR) of the instrument by comparing the difference between the average signal with a sample present and with water as a null reference. This process requires a vial containing 500 uL of 30 uM fluorescein and a vial containing the same amount of water. <br />
<br />
# Run the <code>Basic DNA Melter GUI</code>. <br />
# Place a vial of DI water in your instrument.<br />
# Clear the data and wait 10 seconds.<br />
# Replace the water vial with the DNA of fluorescein vial, record for another 10 seconds, then save the data. <br />
#* Be sure that all other conditions, such as temperature, are stable throughout the test. <br />
# Segment your data into that when the water was present and that when the fluorescein was present. Calculate the average signal in each case. Record the difference between the two averages as your signal. <br />
# Now discard the water portion of your signal and subtract the average fluorescein signal value from the fluorescein signal.<br />
# Measure the standard deviation of this residual and record as record as your estimate of the noise level.<br />
# '''Calculate the SNR as signal divided by noise and record in your lab report.'''<br />
<br />
===Experiment steps===<br />
<br />
Once the instrument is confirmed to be functioning properly, follow these instructions to generate melting curve data for the 20 bp DNA-LC Green solution.<br />
<br />
#Open and run the <code>Basic DNA Melter GUI</code> in Matlab and follow the instructions there.<br />
# Confirm that block temperature is near room temperature when the Diablotek supply is off.<br />
# Apply heat to the block by switching the Diablotek on to increase the block temperature to 95&deg;C, toggle the Diablotek power in order to hold the temperature long enough for the fluorescence curve to reach a minimum, then allow the block to cool down by switching off the Diablotek.<br />
#* Other approaches may be used. One goal would be to heat the block more slowly, possibly by toggling the current while heating.<br />
# Now prepare a sample using the stock of "beater" DNA.<br />
# Place the vial in the instrument, but keep the LED off until ready to measure the melting curve.<br />
# Measure melting curves of the beater DNA sample as-desired to understand instrument operation.<br />
# Prepare a sample using fresh 20 bp 100 mM KCl DNA.<br />
# Measure a melting curve of the fresh sample.<br />
#* Be sure to save a copy in a local directory.<br />
<br />
{{:DNA Melting Report Requirements for Part 1}}<br />
<br />
==Lab manual sections==<br />
<br />
*[[Lab Manual:Measuring DNA Melting Curves]]<br />
*[[DNA Melting: Simulating DNA Melting - Basics]]<br />
*[[DNA Melting Part 1: Measuring Temperature and Fluorescence]]<br />
*[[DNA Melting Report Requirements for Part 1]]<br />
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]<br />
*[[DNA Melting Report Requirements for Part 2]]<br />
<br />
==References==<br />
<references/><br />
<br />
==Subset of datasheets==<br />
(Many more can be found online or on the course share)<br />
#[http://www.ni.com/pdf/manuals/371931f.pdf National Instruments USB-6212 user manual]<br />
#[http://www.ni.com/pdf/manuals/370784d.pdf National Instruments USB-6341 user manual]<br />
#[http://www.national.com/ds/LF/LF411.pdf Op-amp datasheet]<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/DNA_Melting_Part_1:_Measuring_Temperature_and_FluorescenceDNA Melting Part 1: Measuring Temperature and Fluorescence2015-11-04T21:12:48Z<p>Steven Nagle: /* Making a rough measurement of the SNR */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
<br />
[[Image:DNA Melting Block Diagram.jpg|frame|DNA melting apparatus block diagram showing the five functional groups of the instrument.]]<br />
<br />
<br />
''In theory, there is no difference between theory and practice. But, in practice, there is.'' <br />
<br />
''&mdash;[http://en.wikiquote.org/wiki/Jan_L._A._van_de_Snepscheut Jan L. A. van de Snepscheut]/[http://en.wikiquote.org/wiki/Yogi_Berra Yogi Berra]/and or [http://c2.com/cgi/wiki?DifferenceBetweenTheoryAndPractice Chuck Reid]'' <br />
<br />
You will construct a basic DNA melting instrument consisting of the five functional groups shown in the block diagram:<br />
* excitation<br />
* heating<br />
* fluorescent detection<br />
* temperature measurement<br />
* data acquisition and control<br />
<br />
A metal block will hold a sample vial in the excitation light path and transfer heat to the sample. The sample contains a combination of DNA, SYBR Green I and a salt. Recall that SYBR Green I fluoresces when it is bound to double stranded DNA (dsDNA) by a factor of up to 1000x compared to when it is bound to single stranded DNA (ssDNA). Detection of this fluorescence provides a relative measure of the fraction of dsDNA to total DNA, which can be compared to predictions from a thermodynamic model.<br />
<br />
The dye has a peak sensitivity to blue light at 497 nm so a blue LED will be used to illuminate the sample. The dye emits green light with an emission peak at 520 nm. A photodiode will be placed at 90&deg; to the LED source to detect the green light emitted by SYBR Green I. Because LEDs have a fairly broad spectrum, first a moderately narrow band-pass filter will be used to limit the excitation wavelength to between 450 and 490 nm. Then a complimentary long-pass emission filter will be placed between the sample and detector to pass only wavelengths longer than 515 nm. This filter combination, when used with this dye system, eliminates any background signal due to the excitation light.<br />
<br />
The sample block will be heated by a TEC (thermoelectric cooler) and its temperature measured by an embedded RTD (resistance temperature detector). Also, since photodiodes produce a very small amount of current, a high gain amplifier will convert the photodiode current into a measurable voltage. The DAQ (data acquisition card) and PC will digitize the amplified photodiode and RTD signals. A Matlab program is provided to record these fluorescence and temperature signals over time and save the data to a file. The file can then be loaded into Matlab for analysis, or if you prefer, Python or equivalent where the signals will be converted into dsDNA concentration and sample temperature. Finally, the dsDNA sample melting temperature will be estimated from a plot of df/dT vs T (''f'' represents the fraction of total DNA that is in double-stranded form) and from a comparison to the thermodynamic model.<br />
<br />
The functional groups in the block diagram are one of two fundamental frameworks used to describe an instrument. The other sub-divides the instrument into categories that often mirror historical areas of engineering. The former is focused on what will be done, including the overall scientific goals and the physical processes to be measured. The latter is focused on how it will be done, e.g. using optics, electronics or perhaps a practical knowledge of thermal management. A good engineer will constantly be thinking of the instrument in both frameworks. <br />
<br />
==Mechanical subsystem==<br />
<br />
[[Image:DNA_Setup_v2_Iso.png|600 px|thumb|center|DNA Melting System Model]]<br />
<br />
Mechanical support for the heating block, sample and optics will be built on a portable optical breadboard from [http://www.thorlabs.com ThorLabs]. Most of the components in the lab are from this supplier because the selection is large, the quality is quite good and the price reasonable. Although you are free to modify the design as the lab progresses, begin a build from the standard design shown above and demonstrated by the example in the lab. Gather the following Thorlabs components but do not start to assemble.<br />
<br />
* 1 MB1224 optical breadboard<br />
* 2 CL5 clamps (or similar)<br />
* 2 1/4-20 5/8" screws<br />
* 1 sheet metal support bracket<br />
* 4 1/4-20 1/4" screws<br />
<br />
==Thermal subsystem==<br />
The thermal subsystem includes all components to control heat flow into and out of the sample and monitor its temperature. Heating is accomplished using thermal electric coolers (TECs) in reverse. The hot side is placed in direct contact with the heating block, which is in direct contact with the sample vial. The TECs will be powered by manually controlling the Diablotek power supply. To avoid contamination of the sample or perturbation of its temperature, the temperature of a resistive temperature detector (RTD) embedded in the heating block will be monitored as a proxy for the sample temperature. In this part of the lab, air convection will cool down the sample when the Diablotek is switched off. The heat sink will help and the fan may be connected to a triple-output power supply to help, but these are included primarily for use in Part 2.<br />
<br />
===Parts===<br />
Gather the following components from the bins on the Center Cabinets, the DNA Melting drawer at the wet bench or on the East Cabinets:<br />
<br />
[[Image:CenterCabinets.jpg|right|250px|thumb|'''Center Cabinets''' The bins for most parts that you need will be set out on top of these cabinets, roughly in the center of the lab.]]<br />
[[Image:WetBench.jpg|right|250px|thumb|'''Wet Bench''' Supplies for our wet bench can be found in the three drawers at the left of the bench, the drawer left of the sink and on the wall behind and to the left as you face the wet bench. The drawer at the right of the wet bench contains various supplies for the DNA Melting instrument.]]<br />
[[Image:EastCabinets.jpg|right|250px|thumb|'''East Cabinets''' The center shelves of the East Cabinets contain the electronic breadboards, breadboard jumper wire, TEC connectors and TEC power supplies. Often these supplies are laid out on top of these cabinets.]]<br />
<br />
* A metal support bracket, having a fan, heatsink, 2 TEC heaters/coolers, and a heating block attached to it (if they're not attached, you'll need to attach them).<br />
* 3 wire nuts<br />
* 1 Diablotek 250 W power supply<br />
* 1 red/black TEC extension wire assembly<br />
<br />
===Assembly instructions===<br />
<br />
[[Image:TEC_picture.jpg|150 px|thumb|right|TEC showing hot side up when current flows into the red lead]]<br />
<br />
Ensure that the following have been done/attached to your metal bracket:<br />
# Attach the fan on the side of the support bracket using the 6-32 screws and nuts.<br />
# Attach the 2x2 heat sink to the bottom of the support bracket using two 4-40 screws at opposite corners.<br />
# Attach the support bracket to the MB1224 breadboard using 1/4-20 1/4" screws.<br />
# Now place the TECs on a napkin or two, with the red lead extending down and to the ''left'' as shown in the figure.<br />
# Apply a very thin layer of thermal grease to the exposed side of one of the TECs using a wooden stick from the drawer.<br />
#* Use a small amount of grease. Too much is worse than none.<br />
#* Best practice is to apply a baby-pea-sized amount, spread with the stick, smooth and remove excess almost to the point of total removal.<br />
# Now place the second TEC on top of the first.<br />
#* If the two TECs slide freely against each other, then there is too much grease.<br />
#** Separate the TECs and smooth/remove the grease until a modest force is required to slide them.<br />
#* Verify that the colors of the leads match. If not, flip one TEC.<br />
# Connect the black lead of the top TEC to the red lead of the bottom TEC using a wire nut.<br />
# Connect the TEC to the extension wire assembly using a wire nut to join the white wire to the red lead of the top TEC and the black wire to the black lead on the bottom TEC.<br />
# Join the white connector on the assembly to the black 2x2 connector of the Diabloteck.<br />
# Test the TECs by holding the them between thumb and finger while briefly flipping the switch on the Diablotek.<br />
#* One side should immediately heat up while the other immediately cools.<br />
#* The top side should be the hot side when the red wires extend down and to the '''left'''.<br />
#* If the Diablotek immediately shuts off (the fan will stop spinning) then there is a short. Check all connections.<br />
# Once the TEC assembly checks out, butter the bottom side of the assembly and place that side on the heat sink with the leads facing to the right.<br />
# Finally, apply thermal grease to the top side of the TEC assembly, place the heating block on top and anchor to the heat sink using the 1/2" long 4-40 screws and plastic washers.<br />
<br />
==Optical subsystem==<br />
<br />
A good optical subsystem design will be compact, stable and simple. It includes the optical components of the excitation and fluorescence detection functional groups from the block diagram. A good design will efficiently illuminate the sample and collect the maximum possible amount of emitted fluorescent light. There are a variety of ways to construct the apparatus. Use the model above and the example system in the lab as guides.<br />
<br />
The following is a rough set of instructions if you're not clear on how to replicate the example system in the lab. In addition to the following parts, you might want to add a few lenses to collimate your LED onto your sample, and to focus the light coming from your sample onto your photodiode. <br />
<br />
* 2 PH2 post holders<br />
* 2 BA1 post holder bases<br />
* 2 1/4-20 1/4" screws<br />
* 2 1/4-20 5/8" screws<br />
* 2 1/4" washers<br />
* 4 CP02 cage plates<br />
* 2 8-32 1/2" setscrews<br />
* 2 TR2 posts<br />
* 6 ER3 cage rails<br />
* 12 4-40 1/4" setscrews (or similar length)<br />
* 4 SML05 lens tubes<br />
* 4 SM1LTRR retaining rings<br />
* 1 blue LED PCB<br />
* 1 D470/40 excitation filter<br />
* 1 E515LPv2 emission filter<br />
* 1 SM1A6 adapter between SM1 and SM05 threads<br />
* 1 SM05PD1A mounted photodiode<br />
<br />
===Assembly instructions===<br />
<br />
# Attach each PH2 to a BA1 using 1/4" screws.<br />
# Attach two of the CP02 cage plates to two TR2 posts using an 8-32 setscrew.<br />
# Insert the TR2/CP02 assembly into the PH2/BA1 assemblies and lock in place with the TS25H setscrews.<br />
# Insert three ER4 cage rails into each CP02 above and lock in place with SS4S025 set screws or similar.<br />
#* Leave the blank hole on the bottom of the assembly.<br />
# Attach one of the remaining CP02 cage plates on the free end of each assembly above and lock in place.<br />
# Attach each of the above assemblies to the MB1224 using a washer and 1/4-20 screw less than 1" in length.<br />
# Arrange on the MB1224 and adjust CP02 heights as desired.<br />
# Mount the LED PCB in an SML05 lens tube and fix with a retaining ring.<br />
# Mount the excitation and emission filters in each of two lens tubes and fix with retaining ring.<br />
#* The arrow label on the thickness of the filter points ''toward the light source''.<br />
# Thread the SM1A6 adapter into a lens tube and fix with a retaining ring.<br />
# Thread the photodiode into the SM1A6 adapter.<br />
#* Teflon pipe thread tape can be used to hold the photodiode in the desired position.<br />
# Mount each lens tube in the appropriate CP02 cage plates.<br />
# Arrange each the resultant assemblies on the breadboard to most fully illuminate the sample vial and to gather the most of the resultant light on the photodiode.<br />
<br />
==Electronics subsystem==<br />
<br />
The electrical subsystem includes the LED with a current-limiting resistor, the photodiode, the transimpedance amplifier and a temperature detection circuit. The remainder of this section provides an example of each of these systems and instructions to build and test each one. Nevertheless, alternate ideas are welcome. Feel free to discuss with the Instructors.<br />
<br />
===Parts===<br />
<br />
Gather the following components from the electronics drawer to the right of the optics drawers, from the counter top opposite the wet bench and from the cable organizer on the East wall to the right of the white board.<br />
* 1 electronic breadboard<br />
* 1 jump wire kit<br />
* 2 red 3' banana cable or similar<br />
* 1 black 3' banana cable or similar<br />
* individual wire, as-needed from the scrap wire box or the wire spools opposite the wet bench<br />
* 1 62 &Omega; resistor from the center counter top on the South wall<br />
* 1 15 k&Omega; resistor<br />
* other resistors, as-needed for the amplifier<br />
* 1 blue LED from the DNA Melting drawer at the far right of the wet bench<br />
* 2 LF411 op-amps from the electronics drawer<br />
* 4 capacitors between 0.1 and 22 uF from either the electronics drawer or the counter top where the screws are stored.<br />
<br />
===Assembly instructions===<br />
<br />
====LED circuit====<br />
The led circuit consists of a 62 &Omega; resistor in series with a blue, jumbo LED. The circuit will be powered by the 5 volt fixed output of the lab power supply. The 62 &Omega; resistor prevents excessive current from flowing through the LED. Excessive current will let the smoke out of the LED.<br />
<br />
# Build the LED circuit on your electronic breadboard.<br />
#* Don't forget to connect the resistor. Directly connecting the LED to 5 V will blow out the LED.<br />
# Disable the output of the lab power supply.<br />
# Connect the LED circuit to the fixed 5 V output.<br />
<br />
====Temperature detection circuit====<br />
A platinum RTD is embedded inside the heating block. The resistance of the RTD varies with temperature according to the equation ''R<sub>RTD</sub>''=1000 &Omega; + 3.85 ''&theta;'', where ''&theta;'' is the temperature of the RTD in degrees Celsius. The easiest way to make a temperature measurement is to connect the RTD in series with a fixed resistor and a power supply. The output voltage of the resulting divider circuit varies as a function of temperature. <br />
<br />
The maximum current of the RTD is 1 mA. You will use a 15 V supply to the circuit, so the resistor must be at least 14 k&Omega;. Use a 15 k&Omega; resistor.<br />
<br />
# '''Find an equation for ''V<sub>out</sub>'' as a function of temperature. What voltage do you expect given an input voltage of 15 V with the RTD at room temperature.'''<br />
# Turn off the output of the triple-output power supply.<br />
# Connect the RTD to the electronic breadboard.<br />
# '''Measure the resistance of the 15 k&Omega; resistor with a DMM and record the value.'''<br />
# Connect the 15k&Omega; resistor in series with the RTD.<br />
# Set the power supply for '''SERIES''' operation.<br />
# Set the output voltage to 15 V on the supply and make note of its exact value using the DMM.<br />
# Set the current limit to 0.1 A.<br />
# Connect the positive terminal of '''CH1''' to a banana terminal on the breadboard for +15 V and wire it to the top red bus strip for +15 V supply.<br />
# Connect the negative terminal of '''CH2''' to a banana terminal on the breadboard for -15 V.<br />
# Connect the positive terminal of '''CH2''' to the black terminal on the breadboard for a ground reference and wire it to the top blue bus strip.<br />
#* Use three jump wires to minimize the parasitic voltage drop due to the breadboard tie points.<br />
# Connect the +15 V bus strip to the second vertical, red bus strip and wire this bus strip to the top of your 15 k&Omega; resistor.<br />
# Turn on the output of the power supply and measure the voltage across the RTD using the DMM.<br />
# '''What is the measured voltage of the RTD at room temperature and how does it compare to your predictions above?'''<br />
# '''What is the temperature, based on your measured voltage and how does it compare with the clock on the wall (or some better measure)?'''<br />
# '''How is the temperature calculation affected by a 1% change in the 15 V input voltage?'''<br />
<br />
====Photodiode amplifier circuit====<br />
<br />
The current produced by the photodiode detector is a few tens of nanoamperes. This very small current must be amplified to a level where it can be easily measured. The data acquisition system you will use can measure voltages between + and -10 V. Use the two-stage op amp circuit shown below to convert the photodiode current to a voltage.<br />
<br />
<center>[[Image:Part1_TransimpedanceAmplifier.jpg|700 px|thumb|center|Multi-stage photodiode amplification circuit]]</center><br />
<br />
You will have to decide how to distribute the gain between the two op amp stages in the circuit. It is best to put as much gain in early stages of a multi-stage amplifier as possible. However, when the gain of a single stage becomes very high, nonidealities of the op amp become bothersome. The first stage of the amplifier will behave reasonably well as long as the gain is less than about 10<sup>6</sup> &Omega;.<br />
<br />
# '''Analyze the photodiode amplifier circuit. Choose resistor values to achieve an overall gain that will yield an output of about 5 V when the input is 500 nA.'''<br />
# Construct the circuit on your breadboard.<br />
#* Disable the power supply before you build.<br />
#* Do not yet connect the photodiode.<br />
#* Orient the LF411 op-amps so that pin 1, which is marked with a dot on the package, is at the upper left.<br />
# Connect the +/- 15 V bus strips to power the op-amps power pins.<br />
# Connect the vertical blue bus strips to the horizontal blue bus strip.<br />
<br />
=====Test connectivity and basic operation=====<br />
<br />
# Verify that all your component connections are correct and match the design.<br />
# Check that wires and components are properly seated in the breadboard and not suspiciously loose.<br />
#* You can use the ohm-meter function of the digital multi-meter to verify continuity.<br />
# Turn on the power supply and verify that +15V appears on pin 7 and -15V on pin 4.<br />
# '''Short pins 2 and 3 (the V- and V+ inputs to the op-amp) with a small wire and measure the output voltages of IC1 and IC2.'''<br />
#* With the photodiode shorted the current signal is zero, but you may still see a significant offset voltage from 50 to 100 mV at IC1 pin 6.<br />
#* Refer to [http://measure.mit.edu/~20.309/wiki/index.php?title=Real_electronics#Real_op-amps Real Electronics, real op-amps] or ask an instructor to understand and address this non-ideal behavior.<br />
<br />
==Data acquisition hardware subsystem==<br />
<br />
{{:20.309:DAQ System}}<br />
<br />
====Summary of DAQ inputs/outputs====<br />
<br />
A special connector has been prepared for you, connected as shown below. The connections and cable wire colors are also summarized [http://dl.dropbox.com/u/12957607/DAQ_Cable_Assembly_for_DNA_melting_lab.pdf in a memo].<br />
<br />
{| class="wikitable"<br />
|-<br />
! Signal Name<br />
! Signal Location<br />
! Ground Location<br />
! Pin wire color<br />
|-<br />
! DAQ Inputs <br />
|-<br />
! RTD<br />
| AI0+<br />
| AI0-<br />
| +Orange / -Black [lone pair, not with third (red) wire]<br />
|-<br />
! Photodiode <br />
| AI1+ <br />
| AI1-<br />
| +Green / -White<br />
|}<br />
<br />
<center>[[Image:DAQ_Wire_Colors.png|450 px|thumb|center|DAQ Connection Cable]]</center><br />
<br />
==DNAMelter software==<br />
<br />
[[Image:BasicDNAMelterIcon.png|75 px|left]]<br />
Use the <code>Basic DNA Melter GUI</code> program (located on the lab computer desktop) to collect data from the apparatus.<br />
<br />
At the end of an experimental run, use the "Save" button to write the date to a file. The data will be tab-delimited and can be read into Matlab with the <code>load</code> command.<br />
<br />
Documentation for the DNAMelter software is available here: [[DNA Melting: Using the Basic DNAMelter GUI]]<br />
<br />
====If you need to debug the DAQ (skip otherwise!)====<br />
* When debugging problems, it is frequently useful to ascertain whether the data acquisition system is working properly. National Instruments provides an application for this purpose called Measurement and Automation Explorer. Use Measurement and Automation Explorer to verify that the DAQ is properly connected to and communicating with the computer. The program also provides the ability to manually control all of the DAQ system's inputs and outputs.<br />
** Launch Measurement and Automation Explorer by selecting Start->All Applications->National Instruments->Measurement and Automation.<br />
** In the "Configuration" tab, select "Devices and Interfaces" and then "NI-DAQMx Devices." Select "Dev1."<br />
** If Dev 1 does not show up, the problem is that computer cannot communicate with the DAQ system. Ensure that the USB cable is connected to the DAQ system. If the cable is connected and the device still does not show up, unplug the USB cable, count to five, and plug it back in. <br />
** If the DAQ shows up under a different name, disconnect the USB cable. Delete all the "NI USB-6xxx" entries and then reconnect the USB cable. Verify that one entry reappears and that it is called "Dev1." <br />
** Select the "Test Panels" tab to manually control or read signals from the DAQ.<br />
* If you run DNAMelter and there is a data acquisition error, for example, "<code>Data acquisition cannot start!</code>", open Measurement and Automation Explorer and follow the steps above. Run DNAMelter again. If it still doesn't work, grab a beer or ask an instructor for help.<br />
* The DAQ has only one analog-to-digital converter (ADC). A multiplexer circuit sequentially samples voltages from the analog inputs configured in the control software. If any of the inputs is outside the allowable range of +/-10V, all of the inputs may behave erratically. The voltages reported may look reasonable so this can be very misleading. If none of the DAQ readings make sense, ensure that all of the analog voltages are in the allowable range and reset the DAQ by cycling the power of the DAQ (use the switch on the 6341 or disconnect the USB of the 6212).<br />
* If the DAQ gets short-circuited, it may crash. If this happens, Matlab will frequently give a deceptive error message, for example claiming that the limits on the band-pass filter do not satisfy some condition. In this case, correct the connection and cycle the DAQ power.<br />
<br />
==Part 1 measurements==<br />
<br />
Please read this entire section before beginning. Then start at Testing the instrument, with reference back to the sample sections as-needed.<br />
<br />
===Making samples===<br />
<br />
{{Template:Biohazard warning|message=LC Green in DMSO is readily absorbed through skin. Synthetic oligonucleotides may be harmful by inhalation, ingestion, or skin absorption. Wear gloves when handling samples. Wear safety goggles at all times when pipetting the LC Green/DNA samples. Do not create aerosols. The health effects of LC Green have not been thoroughly investigated. See the LC Green and synthetic oligonucleotide under <code>../EHS Guidelines/MSDS Repository</code> in the course locker for more information.}}<br />
<br />
====Sample prep steps====<br />
<br />
#Pipet 500 &mu;L of DNA plus dye solution into a glass vial. <br />
#Pipet up to 200 &mu;L of mineral oil on top of the sample to help prevent evaporation. It will help to pipette into the corners first. Be careful not to get oil on the cuvette sides far above the sample. It will run down during your experiment and cause shifts in the photodiode signal. Place the cap loosely on the vial. <br />
#*Keep the sample vertical to make sure the oil stays on top. <br />
<br />
* You can use the same sample for several heating/cooling cycles.<br />
* Only discard a sample if you lose significant volume due to evaporation or if your signal gets too low. <br />
<br />
To clean your glass vial between samples, flush with alcohol in the waste container and rinse with water at the drain. Suck out residual liquid with the vacuum and drawn Pasteur pipette to the left of the sink.<br />
<br />
====Sample disposal====<br />
<br />
{{Template:Environmental Warning|message=Discard pipette tips with DNA sample residue in the pipette tips or the ''Biohazard Sharps'' container. Do not pour synthetic oligonucleotides with LC Green down the drain. Pour your used samples into the waste container provided in the middle of the wet bench.}}<br />
<br />
===Testing the instrument===<br />
<br />
Once your apparatus is built, use samples of fluorescein (~ 30&mu;M concentration) as a stand-in for LC Green + DNA to test the instrument. Fluorescein is cheap, non-toxic and will not bleach as quickly as LC Green.<br />
<br />
====Making a simple measurement of your instrument's low frequency SNR====<br />
<br />
Carry out a simple estimate of the signal to noise ratio (SNR) of the instrument by comparing the difference between the average signal with a sample present and with water as a null reference. This process requires a vial containing 500 uL of 30 uM fluorescein and a vial containing the same amount of water. <br />
<br />
# Run the <code>Basic DNA Melter GUI</code>. <br />
# Place a vial of DI water in your instrument.<br />
# Clear the data and wait a few seconds. <br />
# Replace the water vial with the DNA of fluorescein vial, record for another few seconds, then save the data. <br />
#* Be sure that all other conditions, such as temperature, are stable throughout the test. <br />
# Segment your data into that when the water was present and that when the fluorescein was present. Calculate the average signal in each case. Record the difference between the two averages as your signal. <br />
# Now discard the water portion of your signal and subtract the average fluorescein signal value from the fluorescein signal.<br />
# Measure the standard deviation of this residual and record as record as your estimate of the noise level.<br />
# '''Calculate the SNR as signal divided by noise and record in your lab report.'''<br />
<br />
===Experiment steps===<br />
<br />
Once the instrument is confirmed to be functioning properly, follow these instructions to generate melting curve data for the 20 bp DNA-LC Green solution.<br />
<br />
#Open and run the <code>Basic DNA Melter GUI</code> in Matlab and follow the instructions there.<br />
# Confirm that block temperature is near room temperature when the Diablotek supply is off.<br />
# Apply heat to the block by switching the Diablotek on to increase the block temperature to 95&deg;C, toggle the Diablotek power in order to hold the temperature long enough for the fluorescence curve to reach a minimum, then allow the block to cool down by switching off the Diablotek.<br />
#* Other approaches may be used. One goal would be to heat the block more slowly, possibly by toggling the current while heating.<br />
# Now prepare a sample using the stock of "beater" DNA.<br />
# Place the vial in the instrument, but keep the LED off until ready to measure the melting curve.<br />
# Measure melting curves of the beater DNA sample as-desired to understand instrument operation.<br />
# Prepare a sample using fresh 20 bp 100 mM KCl DNA.<br />
# Measure a melting curve of the fresh sample.<br />
#* Be sure to save a copy in a local directory.<br />
<br />
{{:DNA Melting Report Requirements for Part 1}}<br />
<br />
==Lab manual sections==<br />
<br />
*[[Lab Manual:Measuring DNA Melting Curves]]<br />
*[[DNA Melting: Simulating DNA Melting - Basics]]<br />
*[[DNA Melting Part 1: Measuring Temperature and Fluorescence]]<br />
*[[DNA Melting Report Requirements for Part 1]]<br />
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]<br />
*[[DNA Melting Report Requirements for Part 2]]<br />
<br />
==References==<br />
<references/><br />
<br />
==Subset of datasheets==<br />
(Many more can be found online or on the course share)<br />
#[http://www.ni.com/pdf/manuals/371931f.pdf National Instruments USB-6212 user manual]<br />
#[http://www.ni.com/pdf/manuals/370784d.pdf National Instruments USB-6341 user manual]<br />
#[http://www.national.com/ds/LF/LF411.pdf Op-amp datasheet]<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/DNA_Melting_Part_1:_Measuring_Temperature_and_FluorescenceDNA Melting Part 1: Measuring Temperature and Fluorescence2015-11-04T20:59:56Z<p>Steven Nagle: /* Test connectivity and basic operation */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
<br />
[[Image:DNA Melting Block Diagram.jpg|frame|DNA melting apparatus block diagram showing the five functional groups of the instrument.]]<br />
<br />
<br />
''In theory, there is no difference between theory and practice. But, in practice, there is.'' <br />
<br />
''&mdash;[http://en.wikiquote.org/wiki/Jan_L._A._van_de_Snepscheut Jan L. A. van de Snepscheut]/[http://en.wikiquote.org/wiki/Yogi_Berra Yogi Berra]/and or [http://c2.com/cgi/wiki?DifferenceBetweenTheoryAndPractice Chuck Reid]'' <br />
<br />
You will construct a basic DNA melting instrument consisting of the five functional groups shown in the block diagram:<br />
* excitation<br />
* heating<br />
* fluorescent detection<br />
* temperature measurement<br />
* data acquisition and control<br />
<br />
A metal block will hold a sample vial in the excitation light path and transfer heat to the sample. The sample contains a combination of DNA, SYBR Green I and a salt. Recall that SYBR Green I fluoresces when it is bound to double stranded DNA (dsDNA) by a factor of up to 1000x compared to when it is bound to single stranded DNA (ssDNA). Detection of this fluorescence provides a relative measure of the fraction of dsDNA to total DNA, which can be compared to predictions from a thermodynamic model.<br />
<br />
The dye has a peak sensitivity to blue light at 497 nm so a blue LED will be used to illuminate the sample. The dye emits green light with an emission peak at 520 nm. A photodiode will be placed at 90&deg; to the LED source to detect the green light emitted by SYBR Green I. Because LEDs have a fairly broad spectrum, first a moderately narrow band-pass filter will be used to limit the excitation wavelength to between 450 and 490 nm. Then a complimentary long-pass emission filter will be placed between the sample and detector to pass only wavelengths longer than 515 nm. This filter combination, when used with this dye system, eliminates any background signal due to the excitation light.<br />
<br />
The sample block will be heated by a TEC (thermoelectric cooler) and its temperature measured by an embedded RTD (resistance temperature detector). Also, since photodiodes produce a very small amount of current, a high gain amplifier will convert the photodiode current into a measurable voltage. The DAQ (data acquisition card) and PC will digitize the amplified photodiode and RTD signals. A Matlab program is provided to record these fluorescence and temperature signals over time and save the data to a file. The file can then be loaded into Matlab for analysis, or if you prefer, Python or equivalent where the signals will be converted into dsDNA concentration and sample temperature. Finally, the dsDNA sample melting temperature will be estimated from a plot of df/dT vs T (''f'' represents the fraction of total DNA that is in double-stranded form) and from a comparison to the thermodynamic model.<br />
<br />
The functional groups in the block diagram are one of two fundamental frameworks used to describe an instrument. The other sub-divides the instrument into categories that often mirror historical areas of engineering. The former is focused on what will be done, including the overall scientific goals and the physical processes to be measured. The latter is focused on how it will be done, e.g. using optics, electronics or perhaps a practical knowledge of thermal management. A good engineer will constantly be thinking of the instrument in both frameworks. <br />
<br />
==Mechanical subsystem==<br />
<br />
[[Image:DNA_Setup_v2_Iso.png|600 px|thumb|center|DNA Melting System Model]]<br />
<br />
Mechanical support for the heating block, sample and optics will be built on a portable optical breadboard from [http://www.thorlabs.com ThorLabs]. Most of the components in the lab are from this supplier because the selection is large, the quality is quite good and the price reasonable. Although you are free to modify the design as the lab progresses, begin a build from the standard design shown above and demonstrated by the example in the lab. Gather the following Thorlabs components but do not start to assemble.<br />
<br />
* 1 MB1224 optical breadboard<br />
* 2 CL5 clamps (or similar)<br />
* 2 1/4-20 5/8" screws<br />
* 1 sheet metal support bracket<br />
* 4 1/4-20 1/4" screws<br />
<br />
==Thermal subsystem==<br />
The thermal subsystem includes all components to control heat flow into and out of the sample and monitor its temperature. Heating is accomplished using thermal electric coolers (TECs) in reverse. The hot side is placed in direct contact with the heating block, which is in direct contact with the sample vial. The TECs will be powered by manually controlling the Diablotek power supply. To avoid contamination of the sample or perturbation of its temperature, the temperature of a resistive temperature detector (RTD) embedded in the heating block will be monitored as a proxy for the sample temperature. In this part of the lab, air convection will cool down the sample when the Diablotek is switched off. The heat sink will help and the fan may be connected to a triple-output power supply to help, but these are included primarily for use in Part 2.<br />
<br />
===Parts===<br />
Gather the following components from the bins on the Center Cabinets, the DNA Melting drawer at the wet bench or on the East Cabinets:<br />
<br />
[[Image:CenterCabinets.jpg|right|250px|thumb|'''Center Cabinets''' The bins for most parts that you need will be set out on top of these cabinets, roughly in the center of the lab.]]<br />
[[Image:WetBench.jpg|right|250px|thumb|'''Wet Bench''' Supplies for our wet bench can be found in the three drawers at the left of the bench, the drawer left of the sink and on the wall behind and to the left as you face the wet bench. The drawer at the right of the wet bench contains various supplies for the DNA Melting instrument.]]<br />
[[Image:EastCabinets.jpg|right|250px|thumb|'''East Cabinets''' The center shelves of the East Cabinets contain the electronic breadboards, breadboard jumper wire, TEC connectors and TEC power supplies. Often these supplies are laid out on top of these cabinets.]]<br />
<br />
* A metal support bracket, having a fan, heatsink, 2 TEC heaters/coolers, and a heating block attached to it (if they're not attached, you'll need to attach them).<br />
* 3 wire nuts<br />
* 1 Diablotek 250 W power supply<br />
* 1 red/black TEC extension wire assembly<br />
<br />
===Assembly instructions===<br />
<br />
[[Image:TEC_picture.jpg|150 px|thumb|right|TEC showing hot side up when current flows into the red lead]]<br />
<br />
Ensure that the following have been done/attached to your metal bracket:<br />
# Attach the fan on the side of the support bracket using the 6-32 screws and nuts.<br />
# Attach the 2x2 heat sink to the bottom of the support bracket using two 4-40 screws at opposite corners.<br />
# Attach the support bracket to the MB1224 breadboard using 1/4-20 1/4" screws.<br />
# Now place the TECs on a napkin or two, with the red lead extending down and to the ''left'' as shown in the figure.<br />
# Apply a very thin layer of thermal grease to the exposed side of one of the TECs using a wooden stick from the drawer.<br />
#* Use a small amount of grease. Too much is worse than none.<br />
#* Best practice is to apply a baby-pea-sized amount, spread with the stick, smooth and remove excess almost to the point of total removal.<br />
# Now place the second TEC on top of the first.<br />
#* If the two TECs slide freely against each other, then there is too much grease.<br />
#** Separate the TECs and smooth/remove the grease until a modest force is required to slide them.<br />
#* Verify that the colors of the leads match. If not, flip one TEC.<br />
# Connect the black lead of the top TEC to the red lead of the bottom TEC using a wire nut.<br />
# Connect the TEC to the extension wire assembly using a wire nut to join the white wire to the red lead of the top TEC and the black wire to the black lead on the bottom TEC.<br />
# Join the white connector on the assembly to the black 2x2 connector of the Diabloteck.<br />
# Test the TECs by holding the them between thumb and finger while briefly flipping the switch on the Diablotek.<br />
#* One side should immediately heat up while the other immediately cools.<br />
#* The top side should be the hot side when the red wires extend down and to the '''left'''.<br />
#* If the Diablotek immediately shuts off (the fan will stop spinning) then there is a short. Check all connections.<br />
# Once the TEC assembly checks out, butter the bottom side of the assembly and place that side on the heat sink with the leads facing to the right.<br />
# Finally, apply thermal grease to the top side of the TEC assembly, place the heating block on top and anchor to the heat sink using the 1/2" long 4-40 screws and plastic washers.<br />
<br />
==Optical subsystem==<br />
<br />
A good optical subsystem design will be compact, stable and simple. It includes the optical components of the excitation and fluorescence detection functional groups from the block diagram. A good design will efficiently illuminate the sample and collect the maximum possible amount of emitted fluorescent light. There are a variety of ways to construct the apparatus. Use the model above and the example system in the lab as guides.<br />
<br />
The following is a rough set of instructions if you're not clear on how to replicate the example system in the lab. In addition to the following parts, you might want to add a few lenses to collimate your LED onto your sample, and to focus the light coming from your sample onto your photodiode. <br />
<br />
* 2 PH2 post holders<br />
* 2 BA1 post holder bases<br />
* 2 1/4-20 1/4" screws<br />
* 2 1/4-20 5/8" screws<br />
* 2 1/4" washers<br />
* 4 CP02 cage plates<br />
* 2 8-32 1/2" setscrews<br />
* 2 TR2 posts<br />
* 6 ER3 cage rails<br />
* 12 4-40 1/4" setscrews (or similar length)<br />
* 4 SML05 lens tubes<br />
* 4 SM1LTRR retaining rings<br />
* 1 blue LED PCB<br />
* 1 D470/40 excitation filter<br />
* 1 E515LPv2 emission filter<br />
* 1 SM1A6 adapter between SM1 and SM05 threads<br />
* 1 SM05PD1A mounted photodiode<br />
<br />
===Assembly instructions===<br />
<br />
# Attach each PH2 to a BA1 using 1/4" screws.<br />
# Attach two of the CP02 cage plates to two TR2 posts using an 8-32 setscrew.<br />
# Insert the TR2/CP02 assembly into the PH2/BA1 assemblies and lock in place with the TS25H setscrews.<br />
# Insert three ER4 cage rails into each CP02 above and lock in place with SS4S025 set screws or similar.<br />
#* Leave the blank hole on the bottom of the assembly.<br />
# Attach one of the remaining CP02 cage plates on the free end of each assembly above and lock in place.<br />
# Attach each of the above assemblies to the MB1224 using a washer and 1/4-20 screw less than 1" in length.<br />
# Arrange on the MB1224 and adjust CP02 heights as desired.<br />
# Mount the LED PCB in an SML05 lens tube and fix with a retaining ring.<br />
# Mount the excitation and emission filters in each of two lens tubes and fix with retaining ring.<br />
#* The arrow label on the thickness of the filter points ''toward the light source''.<br />
# Thread the SM1A6 adapter into a lens tube and fix with a retaining ring.<br />
# Thread the photodiode into the SM1A6 adapter.<br />
#* Teflon pipe thread tape can be used to hold the photodiode in the desired position.<br />
# Mount each lens tube in the appropriate CP02 cage plates.<br />
# Arrange each the resultant assemblies on the breadboard to most fully illuminate the sample vial and to gather the most of the resultant light on the photodiode.<br />
<br />
==Electronics subsystem==<br />
<br />
The electrical subsystem includes the LED with a current-limiting resistor, the photodiode, the transimpedance amplifier and a temperature detection circuit. The remainder of this section provides an example of each of these systems and instructions to build and test each one. Nevertheless, alternate ideas are welcome. Feel free to discuss with the Instructors.<br />
<br />
===Parts===<br />
<br />
Gather the following components from the electronics drawer to the right of the optics drawers, from the counter top opposite the wet bench and from the cable organizer on the East wall to the right of the white board.<br />
* 1 electronic breadboard<br />
* 1 jump wire kit<br />
* 2 red 3' banana cable or similar<br />
* 1 black 3' banana cable or similar<br />
* individual wire, as-needed from the scrap wire box or the wire spools opposite the wet bench<br />
* 1 62 &Omega; resistor from the center counter top on the South wall<br />
* 1 15 k&Omega; resistor<br />
* other resistors, as-needed for the amplifier<br />
* 1 blue LED from the DNA Melting drawer at the far right of the wet bench<br />
* 2 LF411 op-amps from the electronics drawer<br />
* 4 capacitors between 0.1 and 22 uF from either the electronics drawer or the counter top where the screws are stored.<br />
<br />
===Assembly instructions===<br />
<br />
====LED circuit====<br />
The led circuit consists of a 62 &Omega; resistor in series with a blue, jumbo LED. The circuit will be powered by the 5 volt fixed output of the lab power supply. The 62 &Omega; resistor prevents excessive current from flowing through the LED. Excessive current will let the smoke out of the LED.<br />
<br />
# Build the LED circuit on your electronic breadboard.<br />
#* Don't forget to connect the resistor. Directly connecting the LED to 5 V will blow out the LED.<br />
# Disable the output of the lab power supply.<br />
# Connect the LED circuit to the fixed 5 V output.<br />
<br />
====Temperature detection circuit====<br />
A platinum RTD is embedded inside the heating block. The resistance of the RTD varies with temperature according to the equation ''R<sub>RTD</sub>''=1000 &Omega; + 3.85 ''&theta;'', where ''&theta;'' is the temperature of the RTD in degrees Celsius. The easiest way to make a temperature measurement is to connect the RTD in series with a fixed resistor and a power supply. The output voltage of the resulting divider circuit varies as a function of temperature. <br />
<br />
The maximum current of the RTD is 1 mA. You will use a 15 V supply to the circuit, so the resistor must be at least 14 k&Omega;. Use a 15 k&Omega; resistor.<br />
<br />
# '''Find an equation for ''V<sub>out</sub>'' as a function of temperature. What voltage do you expect given an input voltage of 15 V with the RTD at room temperature.'''<br />
# Turn off the output of the triple-output power supply.<br />
# Connect the RTD to the electronic breadboard.<br />
# '''Measure the resistance of the 15 k&Omega; resistor with a DMM and record the value.'''<br />
# Connect the 15k&Omega; resistor in series with the RTD.<br />
# Set the power supply for '''SERIES''' operation.<br />
# Set the output voltage to 15 V on the supply and make note of its exact value using the DMM.<br />
# Set the current limit to 0.1 A.<br />
# Connect the positive terminal of '''CH1''' to a banana terminal on the breadboard for +15 V and wire it to the top red bus strip for +15 V supply.<br />
# Connect the negative terminal of '''CH2''' to a banana terminal on the breadboard for -15 V.<br />
# Connect the positive terminal of '''CH2''' to the black terminal on the breadboard for a ground reference and wire it to the top blue bus strip.<br />
#* Use three jump wires to minimize the parasitic voltage drop due to the breadboard tie points.<br />
# Connect the +15 V bus strip to the second vertical, red bus strip and wire this bus strip to the top of your 15 k&Omega; resistor.<br />
# Turn on the output of the power supply and measure the voltage across the RTD using the DMM.<br />
# '''What is the measured voltage of the RTD at room temperature and how does it compare to your predictions above?'''<br />
# '''What is the temperature, based on your measured voltage and how does it compare with the clock on the wall (or some better measure)?'''<br />
# '''How is the temperature calculation affected by a 1% change in the 15 V input voltage?'''<br />
<br />
====Photodiode amplifier circuit====<br />
<br />
The current produced by the photodiode detector is a few tens of nanoamperes. This very small current must be amplified to a level where it can be easily measured. The data acquisition system you will use can measure voltages between + and -10 V. Use the two-stage op amp circuit shown below to convert the photodiode current to a voltage.<br />
<br />
<center>[[Image:Part1_TransimpedanceAmplifier.jpg|700 px|thumb|center|Multi-stage photodiode amplification circuit]]</center><br />
<br />
You will have to decide how to distribute the gain between the two op amp stages in the circuit. It is best to put as much gain in early stages of a multi-stage amplifier as possible. However, when the gain of a single stage becomes very high, nonidealities of the op amp become bothersome. The first stage of the amplifier will behave reasonably well as long as the gain is less than about 10<sup>6</sup> &Omega;.<br />
<br />
# '''Analyze the photodiode amplifier circuit. Choose resistor values to achieve an overall gain that will yield an output of about 5 V when the input is 500 nA.'''<br />
# Construct the circuit on your breadboard.<br />
#* Disable the power supply before you build.<br />
#* Do not yet connect the photodiode.<br />
#* Orient the LF411 op-amps so that pin 1, which is marked with a dot on the package, is at the upper left.<br />
# Connect the +/- 15 V bus strips to power the op-amps power pins.<br />
# Connect the vertical blue bus strips to the horizontal blue bus strip.<br />
<br />
=====Test connectivity and basic operation=====<br />
<br />
# Verify that all your component connections are correct and match the design.<br />
# Check that wires and components are properly seated in the breadboard and not suspiciously loose.<br />
#* You can use the ohm-meter function of the digital multi-meter to verify continuity.<br />
# Turn on the power supply and verify that +15V appears on pin 7 and -15V on pin 4.<br />
# '''Short pins 2 and 3 (the V- and V+ inputs to the op-amp) with a small wire and measure the output voltages of IC1 and IC2.'''<br />
#* With the photodiode shorted the current signal is zero, but you may still see a significant offset voltage from 50 to 100 mV at IC1 pin 6.<br />
#* Refer to [http://measure.mit.edu/~20.309/wiki/index.php?title=Real_electronics#Real_op-amps Real Electronics, real op-amps] or ask an instructor to understand and address this non-ideal behavior.<br />
<br />
==Data acquisition hardware subsystem==<br />
<br />
{{:20.309:DAQ System}}<br />
<br />
====Summary of DAQ inputs/outputs====<br />
<br />
A special connector has been prepared for you, connected as shown below. The connections and cable wire colors are also summarized [http://dl.dropbox.com/u/12957607/DAQ_Cable_Assembly_for_DNA_melting_lab.pdf in a memo].<br />
<br />
{| class="wikitable"<br />
|-<br />
! Signal Name<br />
! Signal Location<br />
! Ground Location<br />
! Pin wire color<br />
|-<br />
! DAQ Inputs <br />
|-<br />
! RTD<br />
| AI0+<br />
| AI0-<br />
| +Orange / -Black [lone pair, not with third (red) wire]<br />
|-<br />
! Photodiode <br />
| AI1+ <br />
| AI1-<br />
| +Green / -White<br />
|}<br />
<br />
<center>[[Image:DAQ_Wire_Colors.png|450 px|thumb|center|DAQ Connection Cable]]</center><br />
<br />
==DNAMelter software==<br />
<br />
[[Image:BasicDNAMelterIcon.png|75 px|left]]<br />
Use the <code>Basic DNA Melter GUI</code> program (located on the lab computer desktop) to collect data from the apparatus.<br />
<br />
At the end of an experimental run, use the "Save" button to write the date to a file. The data will be tab-delimited and can be read into Matlab with the <code>load</code> command.<br />
<br />
Documentation for the DNAMelter software is available here: [[DNA Melting: Using the Basic DNAMelter GUI]]<br />
<br />
====If you need to debug the DAQ (skip otherwise!)====<br />
* When debugging problems, it is frequently useful to ascertain whether the data acquisition system is working properly. National Instruments provides an application for this purpose called Measurement and Automation Explorer. Use Measurement and Automation Explorer to verify that the DAQ is properly connected to and communicating with the computer. The program also provides the ability to manually control all of the DAQ system's inputs and outputs.<br />
** Launch Measurement and Automation Explorer by selecting Start->All Applications->National Instruments->Measurement and Automation.<br />
** In the "Configuration" tab, select "Devices and Interfaces" and then "NI-DAQMx Devices." Select "Dev1."<br />
** If Dev 1 does not show up, the problem is that computer cannot communicate with the DAQ system. Ensure that the USB cable is connected to the DAQ system. If the cable is connected and the device still does not show up, unplug the USB cable, count to five, and plug it back in. <br />
** If the DAQ shows up under a different name, disconnect the USB cable. Delete all the "NI USB-6xxx" entries and then reconnect the USB cable. Verify that one entry reappears and that it is called "Dev1." <br />
** Select the "Test Panels" tab to manually control or read signals from the DAQ.<br />
* If you run DNAMelter and there is a data acquisition error, for example, "<code>Data acquisition cannot start!</code>", open Measurement and Automation Explorer and follow the steps above. Run DNAMelter again. If it still doesn't work, grab a beer or ask an instructor for help.<br />
* The DAQ has only one analog-to-digital converter (ADC). A multiplexer circuit sequentially samples voltages from the analog inputs configured in the control software. If any of the inputs is outside the allowable range of +/-10V, all of the inputs may behave erratically. The voltages reported may look reasonable so this can be very misleading. If none of the DAQ readings make sense, ensure that all of the analog voltages are in the allowable range and reset the DAQ by cycling the power of the DAQ (use the switch on the 6341 or disconnect the USB of the 6212).<br />
* If the DAQ gets short-circuited, it may crash. If this happens, Matlab will frequently give a deceptive error message, for example claiming that the limits on the band-pass filter do not satisfy some condition. In this case, correct the connection and cycle the DAQ power.<br />
<br />
==Part 1 measurements==<br />
<br />
Please read this entire section before beginning. Then start at Testing the instrument, with reference back to the sample sections as-needed.<br />
<br />
===Making samples===<br />
<br />
{{Template:Biohazard warning|message=LC Green in DMSO is readily absorbed through skin. Synthetic oligonucleotides may be harmful by inhalation, ingestion, or skin absorption. Wear gloves when handling samples. Wear safety goggles at all times when pipetting the LC Green/DNA samples. Do not create aerosols. The health effects of LC Green have not been thoroughly investigated. See the LC Green and synthetic oligonucleotide under <code>../EHS Guidelines/MSDS Repository</code> in the course locker for more information.}}<br />
<br />
====Sample prep steps====<br />
<br />
#Pipet 500 &mu;L of DNA plus dye solution into a glass vial. <br />
#Pipet up to 200 &mu;L of mineral oil on top of the sample to help prevent evaporation. It will help to pipette into the corners first. Be careful not to get oil on the cuvette sides far above the sample. It will run down during your experiment and cause shifts in the photodiode signal. Place the cap loosely on the vial. <br />
#*Keep the sample vertical to make sure the oil stays on top. <br />
<br />
* You can use the same sample for several heating/cooling cycles.<br />
* Only discard a sample if you lose significant volume due to evaporation or if your signal gets too low. <br />
<br />
To clean your glass vial between samples, flush with alcohol in the waste container and rinse with water at the drain. Suck out residual liquid with the vacuum and drawn Pasteur pipette to the left of the sink.<br />
<br />
====Sample disposal====<br />
<br />
{{Template:Environmental Warning|message=Discard pipette tips with DNA sample residue in the pipette tips or the ''Biohazard Sharps'' container. Do not pour synthetic oligonucleotides with LC Green down the drain. Pour your used samples into the waste container provided in the middle of the wet bench.}}<br />
<br />
===Testing the instrument===<br />
<br />
Once your apparatus is built, use samples of fluorescein (~ 30&mu;M concentration) as a stand-in for LC Green + DNA to test the instrument. Fluorescein is cheap, non-toxic and will not bleach as quickly as LC Green.<br />
<br />
====Making a rough measurement of the SNR====<br />
<br />
Carry out a simple estimate of the signal to noise ratio (SNR) of the instrument by comparing the difference between the average signal with a sample present and with water as a null reference. Calculate the noise level with no sample present. This process requires 500 uL of 30 uM fluorescein and a vial containing the same amount of water. <br />
<br />
# Use <code>Basic DNA Melter GUI</code> to record the photodiode signal with the water vial in the heating block. <br />
# Start by clearing the data and then wait about 10 s. <br />
# Replace the water vial with the DNA of fluorescein vial and record for another 10 s then save the data. <br />
#* Be sure that all other conditions of the instrument are stable throughout the test. <br />
# Plot this data in Matlab and and compare the average signal levels for the water segment of the data and the fluorescein segment.<br />
#* The difference between the two average signal levels is the "signal" portion of the signal-to-noise ratio (SNR) calculation. <br />
# Now measure the noise. Subtract the average value from the fluorescein portion of the signal. This is the noise in your measurement. Measure the standard deviation of this residual. This is your initial estimate of the noise level.<br />
# Add any estimate of the long-term noise to the Std Dev calculated from the signal. This is the "noise" portion of the SNR.<br />
# '''Calculate the SNR as signal divided by noise.'''<br />
<br />
===Experiment steps===<br />
<br />
Once the instrument is confirmed to be functioning properly, follow these instructions to generate melting curve data for the 20 bp DNA-LC Green solution.<br />
<br />
#Open and run the <code>Basic DNA Melter GUI</code> in Matlab and follow the instructions there.<br />
# Confirm that block temperature is near room temperature when the Diablotek supply is off.<br />
# Apply heat to the block by switching the Diablotek on to increase the block temperature to 95&deg;C, toggle the Diablotek power in order to hold the temperature long enough for the fluorescence curve to reach a minimum, then allow the block to cool down by switching off the Diablotek.<br />
#* Other approaches may be used. One goal would be to heat the block more slowly, possibly by toggling the current while heating.<br />
# Now prepare a sample using the stock of "beater" DNA.<br />
# Place the vial in the instrument, but keep the LED off until ready to measure the melting curve.<br />
# Measure melting curves of the beater DNA sample as-desired to understand instrument operation.<br />
# Prepare a sample using fresh 20 bp 100 mM KCl DNA.<br />
# Measure a melting curve of the fresh sample.<br />
#* Be sure to save a copy in a local directory.<br />
<br />
{{:DNA Melting Report Requirements for Part 1}}<br />
<br />
==Lab manual sections==<br />
<br />
*[[Lab Manual:Measuring DNA Melting Curves]]<br />
*[[DNA Melting: Simulating DNA Melting - Basics]]<br />
*[[DNA Melting Part 1: Measuring Temperature and Fluorescence]]<br />
*[[DNA Melting Report Requirements for Part 1]]<br />
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]<br />
*[[DNA Melting Report Requirements for Part 2]]<br />
<br />
==References==<br />
<references/><br />
<br />
==Subset of datasheets==<br />
(Many more can be found online or on the course share)<br />
#[http://www.ni.com/pdf/manuals/371931f.pdf National Instruments USB-6212 user manual]<br />
#[http://www.ni.com/pdf/manuals/370784d.pdf National Instruments USB-6341 user manual]<br />
#[http://www.national.com/ds/LF/LF411.pdf Op-amp datasheet]<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/DNA_Melting_Part_1:_Measuring_Temperature_and_FluorescenceDNA Melting Part 1: Measuring Temperature and Fluorescence2015-11-04T20:59:26Z<p>Steven Nagle: /* Test connectivity and basic operation */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
<br />
[[Image:DNA Melting Block Diagram.jpg|frame|DNA melting apparatus block diagram showing the five functional groups of the instrument.]]<br />
<br />
<br />
''In theory, there is no difference between theory and practice. But, in practice, there is.'' <br />
<br />
''&mdash;[http://en.wikiquote.org/wiki/Jan_L._A._van_de_Snepscheut Jan L. A. van de Snepscheut]/[http://en.wikiquote.org/wiki/Yogi_Berra Yogi Berra]/and or [http://c2.com/cgi/wiki?DifferenceBetweenTheoryAndPractice Chuck Reid]'' <br />
<br />
You will construct a basic DNA melting instrument consisting of the five functional groups shown in the block diagram:<br />
* excitation<br />
* heating<br />
* fluorescent detection<br />
* temperature measurement<br />
* data acquisition and control<br />
<br />
A metal block will hold a sample vial in the excitation light path and transfer heat to the sample. The sample contains a combination of DNA, SYBR Green I and a salt. Recall that SYBR Green I fluoresces when it is bound to double stranded DNA (dsDNA) by a factor of up to 1000x compared to when it is bound to single stranded DNA (ssDNA). Detection of this fluorescence provides a relative measure of the fraction of dsDNA to total DNA, which can be compared to predictions from a thermodynamic model.<br />
<br />
The dye has a peak sensitivity to blue light at 497 nm so a blue LED will be used to illuminate the sample. The dye emits green light with an emission peak at 520 nm. A photodiode will be placed at 90&deg; to the LED source to detect the green light emitted by SYBR Green I. Because LEDs have a fairly broad spectrum, first a moderately narrow band-pass filter will be used to limit the excitation wavelength to between 450 and 490 nm. Then a complimentary long-pass emission filter will be placed between the sample and detector to pass only wavelengths longer than 515 nm. This filter combination, when used with this dye system, eliminates any background signal due to the excitation light.<br />
<br />
The sample block will be heated by a TEC (thermoelectric cooler) and its temperature measured by an embedded RTD (resistance temperature detector). Also, since photodiodes produce a very small amount of current, a high gain amplifier will convert the photodiode current into a measurable voltage. The DAQ (data acquisition card) and PC will digitize the amplified photodiode and RTD signals. A Matlab program is provided to record these fluorescence and temperature signals over time and save the data to a file. The file can then be loaded into Matlab for analysis, or if you prefer, Python or equivalent where the signals will be converted into dsDNA concentration and sample temperature. Finally, the dsDNA sample melting temperature will be estimated from a plot of df/dT vs T (''f'' represents the fraction of total DNA that is in double-stranded form) and from a comparison to the thermodynamic model.<br />
<br />
The functional groups in the block diagram are one of two fundamental frameworks used to describe an instrument. The other sub-divides the instrument into categories that often mirror historical areas of engineering. The former is focused on what will be done, including the overall scientific goals and the physical processes to be measured. The latter is focused on how it will be done, e.g. using optics, electronics or perhaps a practical knowledge of thermal management. A good engineer will constantly be thinking of the instrument in both frameworks. <br />
<br />
==Mechanical subsystem==<br />
<br />
[[Image:DNA_Setup_v2_Iso.png|600 px|thumb|center|DNA Melting System Model]]<br />
<br />
Mechanical support for the heating block, sample and optics will be built on a portable optical breadboard from [http://www.thorlabs.com ThorLabs]. Most of the components in the lab are from this supplier because the selection is large, the quality is quite good and the price reasonable. Although you are free to modify the design as the lab progresses, begin a build from the standard design shown above and demonstrated by the example in the lab. Gather the following Thorlabs components but do not start to assemble.<br />
<br />
* 1 MB1224 optical breadboard<br />
* 2 CL5 clamps (or similar)<br />
* 2 1/4-20 5/8" screws<br />
* 1 sheet metal support bracket<br />
* 4 1/4-20 1/4" screws<br />
<br />
==Thermal subsystem==<br />
The thermal subsystem includes all components to control heat flow into and out of the sample and monitor its temperature. Heating is accomplished using thermal electric coolers (TECs) in reverse. The hot side is placed in direct contact with the heating block, which is in direct contact with the sample vial. The TECs will be powered by manually controlling the Diablotek power supply. To avoid contamination of the sample or perturbation of its temperature, the temperature of a resistive temperature detector (RTD) embedded in the heating block will be monitored as a proxy for the sample temperature. In this part of the lab, air convection will cool down the sample when the Diablotek is switched off. The heat sink will help and the fan may be connected to a triple-output power supply to help, but these are included primarily for use in Part 2.<br />
<br />
===Parts===<br />
Gather the following components from the bins on the Center Cabinets, the DNA Melting drawer at the wet bench or on the East Cabinets:<br />
<br />
[[Image:CenterCabinets.jpg|right|250px|thumb|'''Center Cabinets''' The bins for most parts that you need will be set out on top of these cabinets, roughly in the center of the lab.]]<br />
[[Image:WetBench.jpg|right|250px|thumb|'''Wet Bench''' Supplies for our wet bench can be found in the three drawers at the left of the bench, the drawer left of the sink and on the wall behind and to the left as you face the wet bench. The drawer at the right of the wet bench contains various supplies for the DNA Melting instrument.]]<br />
[[Image:EastCabinets.jpg|right|250px|thumb|'''East Cabinets''' The center shelves of the East Cabinets contain the electronic breadboards, breadboard jumper wire, TEC connectors and TEC power supplies. Often these supplies are laid out on top of these cabinets.]]<br />
<br />
* A metal support bracket, having a fan, heatsink, 2 TEC heaters/coolers, and a heating block attached to it (if they're not attached, you'll need to attach them).<br />
* 3 wire nuts<br />
* 1 Diablotek 250 W power supply<br />
* 1 red/black TEC extension wire assembly<br />
<br />
===Assembly instructions===<br />
<br />
[[Image:TEC_picture.jpg|150 px|thumb|right|TEC showing hot side up when current flows into the red lead]]<br />
<br />
Ensure that the following have been done/attached to your metal bracket:<br />
# Attach the fan on the side of the support bracket using the 6-32 screws and nuts.<br />
# Attach the 2x2 heat sink to the bottom of the support bracket using two 4-40 screws at opposite corners.<br />
# Attach the support bracket to the MB1224 breadboard using 1/4-20 1/4" screws.<br />
# Now place the TECs on a napkin or two, with the red lead extending down and to the ''left'' as shown in the figure.<br />
# Apply a very thin layer of thermal grease to the exposed side of one of the TECs using a wooden stick from the drawer.<br />
#* Use a small amount of grease. Too much is worse than none.<br />
#* Best practice is to apply a baby-pea-sized amount, spread with the stick, smooth and remove excess almost to the point of total removal.<br />
# Now place the second TEC on top of the first.<br />
#* If the two TECs slide freely against each other, then there is too much grease.<br />
#** Separate the TECs and smooth/remove the grease until a modest force is required to slide them.<br />
#* Verify that the colors of the leads match. If not, flip one TEC.<br />
# Connect the black lead of the top TEC to the red lead of the bottom TEC using a wire nut.<br />
# Connect the TEC to the extension wire assembly using a wire nut to join the white wire to the red lead of the top TEC and the black wire to the black lead on the bottom TEC.<br />
# Join the white connector on the assembly to the black 2x2 connector of the Diabloteck.<br />
# Test the TECs by holding the them between thumb and finger while briefly flipping the switch on the Diablotek.<br />
#* One side should immediately heat up while the other immediately cools.<br />
#* The top side should be the hot side when the red wires extend down and to the '''left'''.<br />
#* If the Diablotek immediately shuts off (the fan will stop spinning) then there is a short. Check all connections.<br />
# Once the TEC assembly checks out, butter the bottom side of the assembly and place that side on the heat sink with the leads facing to the right.<br />
# Finally, apply thermal grease to the top side of the TEC assembly, place the heating block on top and anchor to the heat sink using the 1/2" long 4-40 screws and plastic washers.<br />
<br />
==Optical subsystem==<br />
<br />
A good optical subsystem design will be compact, stable and simple. It includes the optical components of the excitation and fluorescence detection functional groups from the block diagram. A good design will efficiently illuminate the sample and collect the maximum possible amount of emitted fluorescent light. There are a variety of ways to construct the apparatus. Use the model above and the example system in the lab as guides.<br />
<br />
The following is a rough set of instructions if you're not clear on how to replicate the example system in the lab. In addition to the following parts, you might want to add a few lenses to collimate your LED onto your sample, and to focus the light coming from your sample onto your photodiode. <br />
<br />
* 2 PH2 post holders<br />
* 2 BA1 post holder bases<br />
* 2 1/4-20 1/4" screws<br />
* 2 1/4-20 5/8" screws<br />
* 2 1/4" washers<br />
* 4 CP02 cage plates<br />
* 2 8-32 1/2" setscrews<br />
* 2 TR2 posts<br />
* 6 ER3 cage rails<br />
* 12 4-40 1/4" setscrews (or similar length)<br />
* 4 SML05 lens tubes<br />
* 4 SM1LTRR retaining rings<br />
* 1 blue LED PCB<br />
* 1 D470/40 excitation filter<br />
* 1 E515LPv2 emission filter<br />
* 1 SM1A6 adapter between SM1 and SM05 threads<br />
* 1 SM05PD1A mounted photodiode<br />
<br />
===Assembly instructions===<br />
<br />
# Attach each PH2 to a BA1 using 1/4" screws.<br />
# Attach two of the CP02 cage plates to two TR2 posts using an 8-32 setscrew.<br />
# Insert the TR2/CP02 assembly into the PH2/BA1 assemblies and lock in place with the TS25H setscrews.<br />
# Insert three ER4 cage rails into each CP02 above and lock in place with SS4S025 set screws or similar.<br />
#* Leave the blank hole on the bottom of the assembly.<br />
# Attach one of the remaining CP02 cage plates on the free end of each assembly above and lock in place.<br />
# Attach each of the above assemblies to the MB1224 using a washer and 1/4-20 screw less than 1" in length.<br />
# Arrange on the MB1224 and adjust CP02 heights as desired.<br />
# Mount the LED PCB in an SML05 lens tube and fix with a retaining ring.<br />
# Mount the excitation and emission filters in each of two lens tubes and fix with retaining ring.<br />
#* The arrow label on the thickness of the filter points ''toward the light source''.<br />
# Thread the SM1A6 adapter into a lens tube and fix with a retaining ring.<br />
# Thread the photodiode into the SM1A6 adapter.<br />
#* Teflon pipe thread tape can be used to hold the photodiode in the desired position.<br />
# Mount each lens tube in the appropriate CP02 cage plates.<br />
# Arrange each the resultant assemblies on the breadboard to most fully illuminate the sample vial and to gather the most of the resultant light on the photodiode.<br />
<br />
==Electronics subsystem==<br />
<br />
The electrical subsystem includes the LED with a current-limiting resistor, the photodiode, the transimpedance amplifier and a temperature detection circuit. The remainder of this section provides an example of each of these systems and instructions to build and test each one. Nevertheless, alternate ideas are welcome. Feel free to discuss with the Instructors.<br />
<br />
===Parts===<br />
<br />
Gather the following components from the electronics drawer to the right of the optics drawers, from the counter top opposite the wet bench and from the cable organizer on the East wall to the right of the white board.<br />
* 1 electronic breadboard<br />
* 1 jump wire kit<br />
* 2 red 3' banana cable or similar<br />
* 1 black 3' banana cable or similar<br />
* individual wire, as-needed from the scrap wire box or the wire spools opposite the wet bench<br />
* 1 62 &Omega; resistor from the center counter top on the South wall<br />
* 1 15 k&Omega; resistor<br />
* other resistors, as-needed for the amplifier<br />
* 1 blue LED from the DNA Melting drawer at the far right of the wet bench<br />
* 2 LF411 op-amps from the electronics drawer<br />
* 4 capacitors between 0.1 and 22 uF from either the electronics drawer or the counter top where the screws are stored.<br />
<br />
===Assembly instructions===<br />
<br />
====LED circuit====<br />
The led circuit consists of a 62 &Omega; resistor in series with a blue, jumbo LED. The circuit will be powered by the 5 volt fixed output of the lab power supply. The 62 &Omega; resistor prevents excessive current from flowing through the LED. Excessive current will let the smoke out of the LED.<br />
<br />
# Build the LED circuit on your electronic breadboard.<br />
#* Don't forget to connect the resistor. Directly connecting the LED to 5 V will blow out the LED.<br />
# Disable the output of the lab power supply.<br />
# Connect the LED circuit to the fixed 5 V output.<br />
<br />
====Temperature detection circuit====<br />
A platinum RTD is embedded inside the heating block. The resistance of the RTD varies with temperature according to the equation ''R<sub>RTD</sub>''=1000 &Omega; + 3.85 ''&theta;'', where ''&theta;'' is the temperature of the RTD in degrees Celsius. The easiest way to make a temperature measurement is to connect the RTD in series with a fixed resistor and a power supply. The output voltage of the resulting divider circuit varies as a function of temperature. <br />
<br />
The maximum current of the RTD is 1 mA. You will use a 15 V supply to the circuit, so the resistor must be at least 14 k&Omega;. Use a 15 k&Omega; resistor.<br />
<br />
# '''Find an equation for ''V<sub>out</sub>'' as a function of temperature. What voltage do you expect given an input voltage of 15 V with the RTD at room temperature.'''<br />
# Turn off the output of the triple-output power supply.<br />
# Connect the RTD to the electronic breadboard.<br />
# '''Measure the resistance of the 15 k&Omega; resistor with a DMM and record the value.'''<br />
# Connect the 15k&Omega; resistor in series with the RTD.<br />
# Set the power supply for '''SERIES''' operation.<br />
# Set the output voltage to 15 V on the supply and make note of its exact value using the DMM.<br />
# Set the current limit to 0.1 A.<br />
# Connect the positive terminal of '''CH1''' to a banana terminal on the breadboard for +15 V and wire it to the top red bus strip for +15 V supply.<br />
# Connect the negative terminal of '''CH2''' to a banana terminal on the breadboard for -15 V.<br />
# Connect the positive terminal of '''CH2''' to the black terminal on the breadboard for a ground reference and wire it to the top blue bus strip.<br />
#* Use three jump wires to minimize the parasitic voltage drop due to the breadboard tie points.<br />
# Connect the +15 V bus strip to the second vertical, red bus strip and wire this bus strip to the top of your 15 k&Omega; resistor.<br />
# Turn on the output of the power supply and measure the voltage across the RTD using the DMM.<br />
# '''What is the measured voltage of the RTD at room temperature and how does it compare to your predictions above?'''<br />
# '''What is the temperature, based on your measured voltage and how does it compare with the clock on the wall (or some better measure)?'''<br />
# '''How is the temperature calculation affected by a 1% change in the 15 V input voltage?'''<br />
<br />
====Photodiode amplifier circuit====<br />
<br />
The current produced by the photodiode detector is a few tens of nanoamperes. This very small current must be amplified to a level where it can be easily measured. The data acquisition system you will use can measure voltages between + and -10 V. Use the two-stage op amp circuit shown below to convert the photodiode current to a voltage.<br />
<br />
<center>[[Image:Part1_TransimpedanceAmplifier.jpg|700 px|thumb|center|Multi-stage photodiode amplification circuit]]</center><br />
<br />
You will have to decide how to distribute the gain between the two op amp stages in the circuit. It is best to put as much gain in early stages of a multi-stage amplifier as possible. However, when the gain of a single stage becomes very high, nonidealities of the op amp become bothersome. The first stage of the amplifier will behave reasonably well as long as the gain is less than about 10<sup>6</sup> &Omega;.<br />
<br />
# '''Analyze the photodiode amplifier circuit. Choose resistor values to achieve an overall gain that will yield an output of about 5 V when the input is 500 nA.'''<br />
# Construct the circuit on your breadboard.<br />
#* Disable the power supply before you build.<br />
#* Do not yet connect the photodiode.<br />
#* Orient the LF411 op-amps so that pin 1, which is marked with a dot on the package, is at the upper left.<br />
# Connect the +/- 15 V bus strips to power the op-amps power pins.<br />
# Connect the vertical blue bus strips to the horizontal blue bus strip.<br />
<br />
=====Test connectivity and basic operation=====<br />
<br />
# Verify that all your component connections are correct and match the design.<br />
# Check that wires and components are properly seated in the breadboard and not suspiciously loose.<br />
#* You can use the ohm-meter function of the digital multi-meter to verify continuity.<br />
# Turn on the power supply and verify that +15V appears on pin 7 and -15V on pin 4.<br />
# Short pins 2 and 3 (the V- and V+ inputs to the op-amp) with a small wire.<br />
# '''Measure the output voltages of IC1 and IC2.'''<br />
#* With the photodiode shorted the current signal is zero, but you may still see a significant offset voltage from 50 to 100 mV at IC1 pin 6.<br />
#* Refer to [http://measure.mit.edu/~20.309/wiki/index.php?title=Real_electronics#Real_op-amps Real Electronics, real op-amps] or ask an instructor to understand and address this non-ideal behavior.<br />
<br />
==Data acquisition hardware subsystem==<br />
<br />
{{:20.309:DAQ System}}<br />
<br />
====Summary of DAQ inputs/outputs====<br />
<br />
A special connector has been prepared for you, connected as shown below. The connections and cable wire colors are also summarized [http://dl.dropbox.com/u/12957607/DAQ_Cable_Assembly_for_DNA_melting_lab.pdf in a memo].<br />
<br />
{| class="wikitable"<br />
|-<br />
! Signal Name<br />
! Signal Location<br />
! Ground Location<br />
! Pin wire color<br />
|-<br />
! DAQ Inputs <br />
|-<br />
! RTD<br />
| AI0+<br />
| AI0-<br />
| +Orange / -Black [lone pair, not with third (red) wire]<br />
|-<br />
! Photodiode <br />
| AI1+ <br />
| AI1-<br />
| +Green / -White<br />
|}<br />
<br />
<center>[[Image:DAQ_Wire_Colors.png|450 px|thumb|center|DAQ Connection Cable]]</center><br />
<br />
==DNAMelter software==<br />
<br />
[[Image:BasicDNAMelterIcon.png|75 px|left]]<br />
Use the <code>Basic DNA Melter GUI</code> program (located on the lab computer desktop) to collect data from the apparatus.<br />
<br />
At the end of an experimental run, use the "Save" button to write the date to a file. The data will be tab-delimited and can be read into Matlab with the <code>load</code> command.<br />
<br />
Documentation for the DNAMelter software is available here: [[DNA Melting: Using the Basic DNAMelter GUI]]<br />
<br />
====If you need to debug the DAQ (skip otherwise!)====<br />
* When debugging problems, it is frequently useful to ascertain whether the data acquisition system is working properly. National Instruments provides an application for this purpose called Measurement and Automation Explorer. Use Measurement and Automation Explorer to verify that the DAQ is properly connected to and communicating with the computer. The program also provides the ability to manually control all of the DAQ system's inputs and outputs.<br />
** Launch Measurement and Automation Explorer by selecting Start->All Applications->National Instruments->Measurement and Automation.<br />
** In the "Configuration" tab, select "Devices and Interfaces" and then "NI-DAQMx Devices." Select "Dev1."<br />
** If Dev 1 does not show up, the problem is that computer cannot communicate with the DAQ system. Ensure that the USB cable is connected to the DAQ system. If the cable is connected and the device still does not show up, unplug the USB cable, count to five, and plug it back in. <br />
** If the DAQ shows up under a different name, disconnect the USB cable. Delete all the "NI USB-6xxx" entries and then reconnect the USB cable. Verify that one entry reappears and that it is called "Dev1." <br />
** Select the "Test Panels" tab to manually control or read signals from the DAQ.<br />
* If you run DNAMelter and there is a data acquisition error, for example, "<code>Data acquisition cannot start!</code>", open Measurement and Automation Explorer and follow the steps above. Run DNAMelter again. If it still doesn't work, grab a beer or ask an instructor for help.<br />
* The DAQ has only one analog-to-digital converter (ADC). A multiplexer circuit sequentially samples voltages from the analog inputs configured in the control software. If any of the inputs is outside the allowable range of +/-10V, all of the inputs may behave erratically. The voltages reported may look reasonable so this can be very misleading. If none of the DAQ readings make sense, ensure that all of the analog voltages are in the allowable range and reset the DAQ by cycling the power of the DAQ (use the switch on the 6341 or disconnect the USB of the 6212).<br />
* If the DAQ gets short-circuited, it may crash. If this happens, Matlab will frequently give a deceptive error message, for example claiming that the limits on the band-pass filter do not satisfy some condition. In this case, correct the connection and cycle the DAQ power.<br />
<br />
==Part 1 measurements==<br />
<br />
Please read this entire section before beginning. Then start at Testing the instrument, with reference back to the sample sections as-needed.<br />
<br />
===Making samples===<br />
<br />
{{Template:Biohazard warning|message=LC Green in DMSO is readily absorbed through skin. Synthetic oligonucleotides may be harmful by inhalation, ingestion, or skin absorption. Wear gloves when handling samples. Wear safety goggles at all times when pipetting the LC Green/DNA samples. Do not create aerosols. The health effects of LC Green have not been thoroughly investigated. See the LC Green and synthetic oligonucleotide under <code>../EHS Guidelines/MSDS Repository</code> in the course locker for more information.}}<br />
<br />
====Sample prep steps====<br />
<br />
#Pipet 500 &mu;L of DNA plus dye solution into a glass vial. <br />
#Pipet up to 200 &mu;L of mineral oil on top of the sample to help prevent evaporation. It will help to pipette into the corners first. Be careful not to get oil on the cuvette sides far above the sample. It will run down during your experiment and cause shifts in the photodiode signal. Place the cap loosely on the vial. <br />
#*Keep the sample vertical to make sure the oil stays on top. <br />
<br />
* You can use the same sample for several heating/cooling cycles.<br />
* Only discard a sample if you lose significant volume due to evaporation or if your signal gets too low. <br />
<br />
To clean your glass vial between samples, flush with alcohol in the waste container and rinse with water at the drain. Suck out residual liquid with the vacuum and drawn Pasteur pipette to the left of the sink.<br />
<br />
====Sample disposal====<br />
<br />
{{Template:Environmental Warning|message=Discard pipette tips with DNA sample residue in the pipette tips or the ''Biohazard Sharps'' container. Do not pour synthetic oligonucleotides with LC Green down the drain. Pour your used samples into the waste container provided in the middle of the wet bench.}}<br />
<br />
===Testing the instrument===<br />
<br />
Once your apparatus is built, use samples of fluorescein (~ 30&mu;M concentration) as a stand-in for LC Green + DNA to test the instrument. Fluorescein is cheap, non-toxic and will not bleach as quickly as LC Green.<br />
<br />
====Making a rough measurement of the SNR====<br />
<br />
Carry out a simple estimate of the signal to noise ratio (SNR) of the instrument by comparing the difference between the average signal with a sample present and with water as a null reference. Calculate the noise level with no sample present. This process requires 500 uL of 30 uM fluorescein and a vial containing the same amount of water. <br />
<br />
# Use <code>Basic DNA Melter GUI</code> to record the photodiode signal with the water vial in the heating block. <br />
# Start by clearing the data and then wait about 10 s. <br />
# Replace the water vial with the DNA of fluorescein vial and record for another 10 s then save the data. <br />
#* Be sure that all other conditions of the instrument are stable throughout the test. <br />
# Plot this data in Matlab and and compare the average signal levels for the water segment of the data and the fluorescein segment.<br />
#* The difference between the two average signal levels is the "signal" portion of the signal-to-noise ratio (SNR) calculation. <br />
# Now measure the noise. Subtract the average value from the fluorescein portion of the signal. This is the noise in your measurement. Measure the standard deviation of this residual. This is your initial estimate of the noise level.<br />
# Add any estimate of the long-term noise to the Std Dev calculated from the signal. This is the "noise" portion of the SNR.<br />
# '''Calculate the SNR as signal divided by noise.'''<br />
<br />
===Experiment steps===<br />
<br />
Once the instrument is confirmed to be functioning properly, follow these instructions to generate melting curve data for the 20 bp DNA-LC Green solution.<br />
<br />
#Open and run the <code>Basic DNA Melter GUI</code> in Matlab and follow the instructions there.<br />
# Confirm that block temperature is near room temperature when the Diablotek supply is off.<br />
# Apply heat to the block by switching the Diablotek on to increase the block temperature to 95&deg;C, toggle the Diablotek power in order to hold the temperature long enough for the fluorescence curve to reach a minimum, then allow the block to cool down by switching off the Diablotek.<br />
#* Other approaches may be used. One goal would be to heat the block more slowly, possibly by toggling the current while heating.<br />
# Now prepare a sample using the stock of "beater" DNA.<br />
# Place the vial in the instrument, but keep the LED off until ready to measure the melting curve.<br />
# Measure melting curves of the beater DNA sample as-desired to understand instrument operation.<br />
# Prepare a sample using fresh 20 bp 100 mM KCl DNA.<br />
# Measure a melting curve of the fresh sample.<br />
#* Be sure to save a copy in a local directory.<br />
<br />
{{:DNA Melting Report Requirements for Part 1}}<br />
<br />
==Lab manual sections==<br />
<br />
*[[Lab Manual:Measuring DNA Melting Curves]]<br />
*[[DNA Melting: Simulating DNA Melting - Basics]]<br />
*[[DNA Melting Part 1: Measuring Temperature and Fluorescence]]<br />
*[[DNA Melting Report Requirements for Part 1]]<br />
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]<br />
*[[DNA Melting Report Requirements for Part 2]]<br />
<br />
==References==<br />
<references/><br />
<br />
==Subset of datasheets==<br />
(Many more can be found online or on the course share)<br />
#[http://www.ni.com/pdf/manuals/371931f.pdf National Instruments USB-6212 user manual]<br />
#[http://www.ni.com/pdf/manuals/370784d.pdf National Instruments USB-6341 user manual]<br />
#[http://www.national.com/ds/LF/LF411.pdf Op-amp datasheet]<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/DNA_Melting_Part_1:_Measuring_Temperature_and_FluorescenceDNA Melting Part 1: Measuring Temperature and Fluorescence2015-11-04T20:55:55Z<p>Steven Nagle: /* Temperature detection circuit */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
<br />
[[Image:DNA Melting Block Diagram.jpg|frame|DNA melting apparatus block diagram showing the five functional groups of the instrument.]]<br />
<br />
<br />
''In theory, there is no difference between theory and practice. But, in practice, there is.'' <br />
<br />
''&mdash;[http://en.wikiquote.org/wiki/Jan_L._A._van_de_Snepscheut Jan L. A. van de Snepscheut]/[http://en.wikiquote.org/wiki/Yogi_Berra Yogi Berra]/and or [http://c2.com/cgi/wiki?DifferenceBetweenTheoryAndPractice Chuck Reid]'' <br />
<br />
You will construct a basic DNA melting instrument consisting of the five functional groups shown in the block diagram:<br />
* excitation<br />
* heating<br />
* fluorescent detection<br />
* temperature measurement<br />
* data acquisition and control<br />
<br />
A metal block will hold a sample vial in the excitation light path and transfer heat to the sample. The sample contains a combination of DNA, SYBR Green I and a salt. Recall that SYBR Green I fluoresces when it is bound to double stranded DNA (dsDNA) by a factor of up to 1000x compared to when it is bound to single stranded DNA (ssDNA). Detection of this fluorescence provides a relative measure of the fraction of dsDNA to total DNA, which can be compared to predictions from a thermodynamic model.<br />
<br />
The dye has a peak sensitivity to blue light at 497 nm so a blue LED will be used to illuminate the sample. The dye emits green light with an emission peak at 520 nm. A photodiode will be placed at 90&deg; to the LED source to detect the green light emitted by SYBR Green I. Because LEDs have a fairly broad spectrum, first a moderately narrow band-pass filter will be used to limit the excitation wavelength to between 450 and 490 nm. Then a complimentary long-pass emission filter will be placed between the sample and detector to pass only wavelengths longer than 515 nm. This filter combination, when used with this dye system, eliminates any background signal due to the excitation light.<br />
<br />
The sample block will be heated by a TEC (thermoelectric cooler) and its temperature measured by an embedded RTD (resistance temperature detector). Also, since photodiodes produce a very small amount of current, a high gain amplifier will convert the photodiode current into a measurable voltage. The DAQ (data acquisition card) and PC will digitize the amplified photodiode and RTD signals. A Matlab program is provided to record these fluorescence and temperature signals over time and save the data to a file. The file can then be loaded into Matlab for analysis, or if you prefer, Python or equivalent where the signals will be converted into dsDNA concentration and sample temperature. Finally, the dsDNA sample melting temperature will be estimated from a plot of df/dT vs T (''f'' represents the fraction of total DNA that is in double-stranded form) and from a comparison to the thermodynamic model.<br />
<br />
The functional groups in the block diagram are one of two fundamental frameworks used to describe an instrument. The other sub-divides the instrument into categories that often mirror historical areas of engineering. The former is focused on what will be done, including the overall scientific goals and the physical processes to be measured. The latter is focused on how it will be done, e.g. using optics, electronics or perhaps a practical knowledge of thermal management. A good engineer will constantly be thinking of the instrument in both frameworks. <br />
<br />
==Mechanical subsystem==<br />
<br />
[[Image:DNA_Setup_v2_Iso.png|600 px|thumb|center|DNA Melting System Model]]<br />
<br />
Mechanical support for the heating block, sample and optics will be built on a portable optical breadboard from [http://www.thorlabs.com ThorLabs]. Most of the components in the lab are from this supplier because the selection is large, the quality is quite good and the price reasonable. Although you are free to modify the design as the lab progresses, begin a build from the standard design shown above and demonstrated by the example in the lab. Gather the following Thorlabs components but do not start to assemble.<br />
<br />
* 1 MB1224 optical breadboard<br />
* 2 CL5 clamps (or similar)<br />
* 2 1/4-20 5/8" screws<br />
* 1 sheet metal support bracket<br />
* 4 1/4-20 1/4" screws<br />
<br />
==Thermal subsystem==<br />
The thermal subsystem includes all components to control heat flow into and out of the sample and monitor its temperature. Heating is accomplished using thermal electric coolers (TECs) in reverse. The hot side is placed in direct contact with the heating block, which is in direct contact with the sample vial. The TECs will be powered by manually controlling the Diablotek power supply. To avoid contamination of the sample or perturbation of its temperature, the temperature of a resistive temperature detector (RTD) embedded in the heating block will be monitored as a proxy for the sample temperature. In this part of the lab, air convection will cool down the sample when the Diablotek is switched off. The heat sink will help and the fan may be connected to a triple-output power supply to help, but these are included primarily for use in Part 2.<br />
<br />
===Parts===<br />
Gather the following components from the bins on the Center Cabinets, the DNA Melting drawer at the wet bench or on the East Cabinets:<br />
<br />
[[Image:CenterCabinets.jpg|right|250px|thumb|'''Center Cabinets''' The bins for most parts that you need will be set out on top of these cabinets, roughly in the center of the lab.]]<br />
[[Image:WetBench.jpg|right|250px|thumb|'''Wet Bench''' Supplies for our wet bench can be found in the three drawers at the left of the bench, the drawer left of the sink and on the wall behind and to the left as you face the wet bench. The drawer at the right of the wet bench contains various supplies for the DNA Melting instrument.]]<br />
[[Image:EastCabinets.jpg|right|250px|thumb|'''East Cabinets''' The center shelves of the East Cabinets contain the electronic breadboards, breadboard jumper wire, TEC connectors and TEC power supplies. Often these supplies are laid out on top of these cabinets.]]<br />
<br />
* A metal support bracket, having a fan, heatsink, 2 TEC heaters/coolers, and a heating block attached to it (if they're not attached, you'll need to attach them).<br />
* 3 wire nuts<br />
* 1 Diablotek 250 W power supply<br />
* 1 red/black TEC extension wire assembly<br />
<br />
===Assembly instructions===<br />
<br />
[[Image:TEC_picture.jpg|150 px|thumb|right|TEC showing hot side up when current flows into the red lead]]<br />
<br />
Ensure that the following have been done/attached to your metal bracket:<br />
# Attach the fan on the side of the support bracket using the 6-32 screws and nuts.<br />
# Attach the 2x2 heat sink to the bottom of the support bracket using two 4-40 screws at opposite corners.<br />
# Attach the support bracket to the MB1224 breadboard using 1/4-20 1/4" screws.<br />
# Now place the TECs on a napkin or two, with the red lead extending down and to the ''left'' as shown in the figure.<br />
# Apply a very thin layer of thermal grease to the exposed side of one of the TECs using a wooden stick from the drawer.<br />
#* Use a small amount of grease. Too much is worse than none.<br />
#* Best practice is to apply a baby-pea-sized amount, spread with the stick, smooth and remove excess almost to the point of total removal.<br />
# Now place the second TEC on top of the first.<br />
#* If the two TECs slide freely against each other, then there is too much grease.<br />
#** Separate the TECs and smooth/remove the grease until a modest force is required to slide them.<br />
#* Verify that the colors of the leads match. If not, flip one TEC.<br />
# Connect the black lead of the top TEC to the red lead of the bottom TEC using a wire nut.<br />
# Connect the TEC to the extension wire assembly using a wire nut to join the white wire to the red lead of the top TEC and the black wire to the black lead on the bottom TEC.<br />
# Join the white connector on the assembly to the black 2x2 connector of the Diabloteck.<br />
# Test the TECs by holding the them between thumb and finger while briefly flipping the switch on the Diablotek.<br />
#* One side should immediately heat up while the other immediately cools.<br />
#* The top side should be the hot side when the red wires extend down and to the '''left'''.<br />
#* If the Diablotek immediately shuts off (the fan will stop spinning) then there is a short. Check all connections.<br />
# Once the TEC assembly checks out, butter the bottom side of the assembly and place that side on the heat sink with the leads facing to the right.<br />
# Finally, apply thermal grease to the top side of the TEC assembly, place the heating block on top and anchor to the heat sink using the 1/2" long 4-40 screws and plastic washers.<br />
<br />
==Optical subsystem==<br />
<br />
A good optical subsystem design will be compact, stable and simple. It includes the optical components of the excitation and fluorescence detection functional groups from the block diagram. A good design will efficiently illuminate the sample and collect the maximum possible amount of emitted fluorescent light. There are a variety of ways to construct the apparatus. Use the model above and the example system in the lab as guides.<br />
<br />
The following is a rough set of instructions if you're not clear on how to replicate the example system in the lab. In addition to the following parts, you might want to add a few lenses to collimate your LED onto your sample, and to focus the light coming from your sample onto your photodiode. <br />
<br />
* 2 PH2 post holders<br />
* 2 BA1 post holder bases<br />
* 2 1/4-20 1/4" screws<br />
* 2 1/4-20 5/8" screws<br />
* 2 1/4" washers<br />
* 4 CP02 cage plates<br />
* 2 8-32 1/2" setscrews<br />
* 2 TR2 posts<br />
* 6 ER3 cage rails<br />
* 12 4-40 1/4" setscrews (or similar length)<br />
* 4 SML05 lens tubes<br />
* 4 SM1LTRR retaining rings<br />
* 1 blue LED PCB<br />
* 1 D470/40 excitation filter<br />
* 1 E515LPv2 emission filter<br />
* 1 SM1A6 adapter between SM1 and SM05 threads<br />
* 1 SM05PD1A mounted photodiode<br />
<br />
===Assembly instructions===<br />
<br />
# Attach each PH2 to a BA1 using 1/4" screws.<br />
# Attach two of the CP02 cage plates to two TR2 posts using an 8-32 setscrew.<br />
# Insert the TR2/CP02 assembly into the PH2/BA1 assemblies and lock in place with the TS25H setscrews.<br />
# Insert three ER4 cage rails into each CP02 above and lock in place with SS4S025 set screws or similar.<br />
#* Leave the blank hole on the bottom of the assembly.<br />
# Attach one of the remaining CP02 cage plates on the free end of each assembly above and lock in place.<br />
# Attach each of the above assemblies to the MB1224 using a washer and 1/4-20 screw less than 1" in length.<br />
# Arrange on the MB1224 and adjust CP02 heights as desired.<br />
# Mount the LED PCB in an SML05 lens tube and fix with a retaining ring.<br />
# Mount the excitation and emission filters in each of two lens tubes and fix with retaining ring.<br />
#* The arrow label on the thickness of the filter points ''toward the light source''.<br />
# Thread the SM1A6 adapter into a lens tube and fix with a retaining ring.<br />
# Thread the photodiode into the SM1A6 adapter.<br />
#* Teflon pipe thread tape can be used to hold the photodiode in the desired position.<br />
# Mount each lens tube in the appropriate CP02 cage plates.<br />
# Arrange each the resultant assemblies on the breadboard to most fully illuminate the sample vial and to gather the most of the resultant light on the photodiode.<br />
<br />
==Electronics subsystem==<br />
<br />
The electrical subsystem includes the LED with a current-limiting resistor, the photodiode, the transimpedance amplifier and a temperature detection circuit. The remainder of this section provides an example of each of these systems and instructions to build and test each one. Nevertheless, alternate ideas are welcome. Feel free to discuss with the Instructors.<br />
<br />
===Parts===<br />
<br />
Gather the following components from the electronics drawer to the right of the optics drawers, from the counter top opposite the wet bench and from the cable organizer on the East wall to the right of the white board.<br />
* 1 electronic breadboard<br />
* 1 jump wire kit<br />
* 2 red 3' banana cable or similar<br />
* 1 black 3' banana cable or similar<br />
* individual wire, as-needed from the scrap wire box or the wire spools opposite the wet bench<br />
* 1 62 &Omega; resistor from the center counter top on the South wall<br />
* 1 15 k&Omega; resistor<br />
* other resistors, as-needed for the amplifier<br />
* 1 blue LED from the DNA Melting drawer at the far right of the wet bench<br />
* 2 LF411 op-amps from the electronics drawer<br />
* 4 capacitors between 0.1 and 22 uF from either the electronics drawer or the counter top where the screws are stored.<br />
<br />
===Assembly instructions===<br />
<br />
====LED circuit====<br />
The led circuit consists of a 62 &Omega; resistor in series with a blue, jumbo LED. The circuit will be powered by the 5 volt fixed output of the lab power supply. The 62 &Omega; resistor prevents excessive current from flowing through the LED. Excessive current will let the smoke out of the LED.<br />
<br />
# Build the LED circuit on your electronic breadboard.<br />
#* Don't forget to connect the resistor. Directly connecting the LED to 5 V will blow out the LED.<br />
# Disable the output of the lab power supply.<br />
# Connect the LED circuit to the fixed 5 V output.<br />
<br />
====Temperature detection circuit====<br />
A platinum RTD is embedded inside the heating block. The resistance of the RTD varies with temperature according to the equation ''R<sub>RTD</sub>''=1000 &Omega; + 3.85 ''&theta;'', where ''&theta;'' is the temperature of the RTD in degrees Celsius. The easiest way to make a temperature measurement is to connect the RTD in series with a fixed resistor and a power supply. The output voltage of the resulting divider circuit varies as a function of temperature. <br />
<br />
The maximum current of the RTD is 1 mA. You will use a 15 V supply to the circuit, so the resistor must be at least 14 k&Omega;. Use a 15 k&Omega; resistor.<br />
<br />
# '''Find an equation for ''V<sub>out</sub>'' as a function of temperature. What voltage do you expect given an input voltage of 15 V with the RTD at room temperature.'''<br />
# Turn off the output of the triple-output power supply.<br />
# Connect the RTD to the electronic breadboard.<br />
# '''Measure the resistance of the 15 k&Omega; resistor with a DMM and record the value.'''<br />
# Connect the 15k&Omega; resistor in series with the RTD.<br />
# Set the power supply for '''SERIES''' operation.<br />
# Set the output voltage to 15 V on the supply and make note of its exact value using the DMM.<br />
# Set the current limit to 0.1 A.<br />
# Connect the positive terminal of '''CH1''' to a banana terminal on the breadboard for +15 V and wire it to the top red bus strip for +15 V supply.<br />
# Connect the negative terminal of '''CH2''' to a banana terminal on the breadboard for -15 V.<br />
# Connect the positive terminal of '''CH2''' to the black terminal on the breadboard for a ground reference and wire it to the top blue bus strip.<br />
#* Use three jump wires to minimize the parasitic voltage drop due to the breadboard tie points.<br />
# Connect the +15 V bus strip to the second vertical, red bus strip and wire this bus strip to the top of your 15 k&Omega; resistor.<br />
# Turn on the output of the power supply and measure the voltage across the RTD using the DMM.<br />
# '''What is the measured voltage of the RTD at room temperature and how does it compare to your predictions above?'''<br />
# '''What is the temperature, based on your measured voltage and how does it compare with the clock on the wall (or some better measure)?'''<br />
# '''How is the temperature calculation affected by a 1% change in the 15 V input voltage?'''<br />
<br />
====Photodiode amplifier circuit====<br />
<br />
The current produced by the photodiode detector is a few tens of nanoamperes. This very small current must be amplified to a level where it can be easily measured. The data acquisition system you will use can measure voltages between + and -10 V. Use the two-stage op amp circuit shown below to convert the photodiode current to a voltage.<br />
<br />
<center>[[Image:Part1_TransimpedanceAmplifier.jpg|700 px|thumb|center|Multi-stage photodiode amplification circuit]]</center><br />
<br />
You will have to decide how to distribute the gain between the two op amp stages in the circuit. It is best to put as much gain in early stages of a multi-stage amplifier as possible. However, when the gain of a single stage becomes very high, nonidealities of the op amp become bothersome. The first stage of the amplifier will behave reasonably well as long as the gain is less than about 10<sup>6</sup> &Omega;.<br />
<br />
# '''Analyze the photodiode amplifier circuit. Choose resistor values to achieve an overall gain that will yield an output of about 5 V when the input is 500 nA.'''<br />
# Construct the circuit on your breadboard.<br />
#* Disable the power supply before you build.<br />
#* Do not yet connect the photodiode.<br />
#* Orient the LF411 op-amps so that pin 1, which is marked with a dot on the package, is at the upper left.<br />
# Connect the +/- 15 V bus strips to power the op-amps power pins.<br />
# Connect the vertical blue bus strips to the horizontal blue bus strip.<br />
<br />
=====Test connectivity and basic operation=====<br />
<br />
# Verify that all your component connections are correct and match the design.<br />
# Check that wires and components are properly seated in the breadboard and not suspiciously loose.<br />
#* You can use the ohm-meter function of the digital multi-meter to verify continuity.<br />
# Turn on the power supply and verify that +15V appears on pin 7 and -15V on pin 4.<br />
# Verify that pins 2 and 3 (the V- and V+ inputs to the op-amp) are equal.<br />
# '''Measure the output voltages of IC1 and IC2.'''<br />
#* Without the photodiode, the input current is zero but you may still see a significant offset voltage from 50 to 100 mV at IC1 pin 6.<br />
#* Refer to [http://measure.mit.edu/~20.309/wiki/index.php?title=Real_electronics#Real_op-amps Real Electronics, real op-amps] to understand, as well as to account for, this non-ideal op-amp behavior.<br />
<br />
==Data acquisition hardware subsystem==<br />
<br />
{{:20.309:DAQ System}}<br />
<br />
====Summary of DAQ inputs/outputs====<br />
<br />
A special connector has been prepared for you, connected as shown below. The connections and cable wire colors are also summarized [http://dl.dropbox.com/u/12957607/DAQ_Cable_Assembly_for_DNA_melting_lab.pdf in a memo].<br />
<br />
{| class="wikitable"<br />
|-<br />
! Signal Name<br />
! Signal Location<br />
! Ground Location<br />
! Pin wire color<br />
|-<br />
! DAQ Inputs <br />
|-<br />
! RTD<br />
| AI0+<br />
| AI0-<br />
| +Orange / -Black [lone pair, not with third (red) wire]<br />
|-<br />
! Photodiode <br />
| AI1+ <br />
| AI1-<br />
| +Green / -White<br />
|}<br />
<br />
<center>[[Image:DAQ_Wire_Colors.png|450 px|thumb|center|DAQ Connection Cable]]</center><br />
<br />
==DNAMelter software==<br />
<br />
[[Image:BasicDNAMelterIcon.png|75 px|left]]<br />
Use the <code>Basic DNA Melter GUI</code> program (located on the lab computer desktop) to collect data from the apparatus.<br />
<br />
At the end of an experimental run, use the "Save" button to write the date to a file. The data will be tab-delimited and can be read into Matlab with the <code>load</code> command.<br />
<br />
Documentation for the DNAMelter software is available here: [[DNA Melting: Using the Basic DNAMelter GUI]]<br />
<br />
====If you need to debug the DAQ (skip otherwise!)====<br />
* When debugging problems, it is frequently useful to ascertain whether the data acquisition system is working properly. National Instruments provides an application for this purpose called Measurement and Automation Explorer. Use Measurement and Automation Explorer to verify that the DAQ is properly connected to and communicating with the computer. The program also provides the ability to manually control all of the DAQ system's inputs and outputs.<br />
** Launch Measurement and Automation Explorer by selecting Start->All Applications->National Instruments->Measurement and Automation.<br />
** In the "Configuration" tab, select "Devices and Interfaces" and then "NI-DAQMx Devices." Select "Dev1."<br />
** If Dev 1 does not show up, the problem is that computer cannot communicate with the DAQ system. Ensure that the USB cable is connected to the DAQ system. If the cable is connected and the device still does not show up, unplug the USB cable, count to five, and plug it back in. <br />
** If the DAQ shows up under a different name, disconnect the USB cable. Delete all the "NI USB-6xxx" entries and then reconnect the USB cable. Verify that one entry reappears and that it is called "Dev1." <br />
** Select the "Test Panels" tab to manually control or read signals from the DAQ.<br />
* If you run DNAMelter and there is a data acquisition error, for example, "<code>Data acquisition cannot start!</code>", open Measurement and Automation Explorer and follow the steps above. Run DNAMelter again. If it still doesn't work, grab a beer or ask an instructor for help.<br />
* The DAQ has only one analog-to-digital converter (ADC). A multiplexer circuit sequentially samples voltages from the analog inputs configured in the control software. If any of the inputs is outside the allowable range of +/-10V, all of the inputs may behave erratically. The voltages reported may look reasonable so this can be very misleading. If none of the DAQ readings make sense, ensure that all of the analog voltages are in the allowable range and reset the DAQ by cycling the power of the DAQ (use the switch on the 6341 or disconnect the USB of the 6212).<br />
* If the DAQ gets short-circuited, it may crash. If this happens, Matlab will frequently give a deceptive error message, for example claiming that the limits on the band-pass filter do not satisfy some condition. In this case, correct the connection and cycle the DAQ power.<br />
<br />
==Part 1 measurements==<br />
<br />
Please read this entire section before beginning. Then start at Testing the instrument, with reference back to the sample sections as-needed.<br />
<br />
===Making samples===<br />
<br />
{{Template:Biohazard warning|message=LC Green in DMSO is readily absorbed through skin. Synthetic oligonucleotides may be harmful by inhalation, ingestion, or skin absorption. Wear gloves when handling samples. Wear safety goggles at all times when pipetting the LC Green/DNA samples. Do not create aerosols. The health effects of LC Green have not been thoroughly investigated. See the LC Green and synthetic oligonucleotide under <code>../EHS Guidelines/MSDS Repository</code> in the course locker for more information.}}<br />
<br />
====Sample prep steps====<br />
<br />
#Pipet 500 &mu;L of DNA plus dye solution into a glass vial. <br />
#Pipet up to 200 &mu;L of mineral oil on top of the sample to help prevent evaporation. It will help to pipette into the corners first. Be careful not to get oil on the cuvette sides far above the sample. It will run down during your experiment and cause shifts in the photodiode signal. Place the cap loosely on the vial. <br />
#*Keep the sample vertical to make sure the oil stays on top. <br />
<br />
* You can use the same sample for several heating/cooling cycles.<br />
* Only discard a sample if you lose significant volume due to evaporation or if your signal gets too low. <br />
<br />
To clean your glass vial between samples, flush with alcohol in the waste container and rinse with water at the drain. Suck out residual liquid with the vacuum and drawn Pasteur pipette to the left of the sink.<br />
<br />
====Sample disposal====<br />
<br />
{{Template:Environmental Warning|message=Discard pipette tips with DNA sample residue in the pipette tips or the ''Biohazard Sharps'' container. Do not pour synthetic oligonucleotides with LC Green down the drain. Pour your used samples into the waste container provided in the middle of the wet bench.}}<br />
<br />
===Testing the instrument===<br />
<br />
Once your apparatus is built, use samples of fluorescein (~ 30&mu;M concentration) as a stand-in for LC Green + DNA to test the instrument. Fluorescein is cheap, non-toxic and will not bleach as quickly as LC Green.<br />
<br />
====Making a rough measurement of the SNR====<br />
<br />
Carry out a simple estimate of the signal to noise ratio (SNR) of the instrument by comparing the difference between the average signal with a sample present and with water as a null reference. Calculate the noise level with no sample present. This process requires 500 uL of 30 uM fluorescein and a vial containing the same amount of water. <br />
<br />
# Use <code>Basic DNA Melter GUI</code> to record the photodiode signal with the water vial in the heating block. <br />
# Start by clearing the data and then wait about 10 s. <br />
# Replace the water vial with the DNA of fluorescein vial and record for another 10 s then save the data. <br />
#* Be sure that all other conditions of the instrument are stable throughout the test. <br />
# Plot this data in Matlab and and compare the average signal levels for the water segment of the data and the fluorescein segment.<br />
#* The difference between the two average signal levels is the "signal" portion of the signal-to-noise ratio (SNR) calculation. <br />
# Now measure the noise. Subtract the average value from the fluorescein portion of the signal. This is the noise in your measurement. Measure the standard deviation of this residual. This is your initial estimate of the noise level.<br />
# Add any estimate of the long-term noise to the Std Dev calculated from the signal. This is the "noise" portion of the SNR.<br />
# '''Calculate the SNR as signal divided by noise.'''<br />
<br />
===Experiment steps===<br />
<br />
Once the instrument is confirmed to be functioning properly, follow these instructions to generate melting curve data for the 20 bp DNA-LC Green solution.<br />
<br />
#Open and run the <code>Basic DNA Melter GUI</code> in Matlab and follow the instructions there.<br />
# Confirm that block temperature is near room temperature when the Diablotek supply is off.<br />
# Apply heat to the block by switching the Diablotek on to increase the block temperature to 95&deg;C, toggle the Diablotek power in order to hold the temperature long enough for the fluorescence curve to reach a minimum, then allow the block to cool down by switching off the Diablotek.<br />
#* Other approaches may be used. One goal would be to heat the block more slowly, possibly by toggling the current while heating.<br />
# Now prepare a sample using the stock of "beater" DNA.<br />
# Place the vial in the instrument, but keep the LED off until ready to measure the melting curve.<br />
# Measure melting curves of the beater DNA sample as-desired to understand instrument operation.<br />
# Prepare a sample using fresh 20 bp 100 mM KCl DNA.<br />
# Measure a melting curve of the fresh sample.<br />
#* Be sure to save a copy in a local directory.<br />
<br />
{{:DNA Melting Report Requirements for Part 1}}<br />
<br />
==Lab manual sections==<br />
<br />
*[[Lab Manual:Measuring DNA Melting Curves]]<br />
*[[DNA Melting: Simulating DNA Melting - Basics]]<br />
*[[DNA Melting Part 1: Measuring Temperature and Fluorescence]]<br />
*[[DNA Melting Report Requirements for Part 1]]<br />
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]<br />
*[[DNA Melting Report Requirements for Part 2]]<br />
<br />
==References==<br />
<references/><br />
<br />
==Subset of datasheets==<br />
(Many more can be found online or on the course share)<br />
#[http://www.ni.com/pdf/manuals/371931f.pdf National Instruments USB-6212 user manual]<br />
#[http://www.ni.com/pdf/manuals/370784d.pdf National Instruments USB-6341 user manual]<br />
#[http://www.national.com/ds/LF/LF411.pdf Op-amp datasheet]<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/DNA_Melting_Part_1:_Measuring_Temperature_and_FluorescenceDNA Melting Part 1: Measuring Temperature and Fluorescence2015-11-04T20:54:18Z<p>Steven Nagle: /* LED circuit */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:DNA Melting Lab]]<br />
{{Template:20.309}}<br />
<br />
[[Image:DNA Melting Block Diagram.jpg|frame|DNA melting apparatus block diagram showing the five functional groups of the instrument.]]<br />
<br />
<br />
''In theory, there is no difference between theory and practice. But, in practice, there is.'' <br />
<br />
''&mdash;[http://en.wikiquote.org/wiki/Jan_L._A._van_de_Snepscheut Jan L. A. van de Snepscheut]/[http://en.wikiquote.org/wiki/Yogi_Berra Yogi Berra]/and or [http://c2.com/cgi/wiki?DifferenceBetweenTheoryAndPractice Chuck Reid]'' <br />
<br />
You will construct a basic DNA melting instrument consisting of the five functional groups shown in the block diagram:<br />
* excitation<br />
* heating<br />
* fluorescent detection<br />
* temperature measurement<br />
* data acquisition and control<br />
<br />
A metal block will hold a sample vial in the excitation light path and transfer heat to the sample. The sample contains a combination of DNA, SYBR Green I and a salt. Recall that SYBR Green I fluoresces when it is bound to double stranded DNA (dsDNA) by a factor of up to 1000x compared to when it is bound to single stranded DNA (ssDNA). Detection of this fluorescence provides a relative measure of the fraction of dsDNA to total DNA, which can be compared to predictions from a thermodynamic model.<br />
<br />
The dye has a peak sensitivity to blue light at 497 nm so a blue LED will be used to illuminate the sample. The dye emits green light with an emission peak at 520 nm. A photodiode will be placed at 90&deg; to the LED source to detect the green light emitted by SYBR Green I. Because LEDs have a fairly broad spectrum, first a moderately narrow band-pass filter will be used to limit the excitation wavelength to between 450 and 490 nm. Then a complimentary long-pass emission filter will be placed between the sample and detector to pass only wavelengths longer than 515 nm. This filter combination, when used with this dye system, eliminates any background signal due to the excitation light.<br />
<br />
The sample block will be heated by a TEC (thermoelectric cooler) and its temperature measured by an embedded RTD (resistance temperature detector). Also, since photodiodes produce a very small amount of current, a high gain amplifier will convert the photodiode current into a measurable voltage. The DAQ (data acquisition card) and PC will digitize the amplified photodiode and RTD signals. A Matlab program is provided to record these fluorescence and temperature signals over time and save the data to a file. The file can then be loaded into Matlab for analysis, or if you prefer, Python or equivalent where the signals will be converted into dsDNA concentration and sample temperature. Finally, the dsDNA sample melting temperature will be estimated from a plot of df/dT vs T (''f'' represents the fraction of total DNA that is in double-stranded form) and from a comparison to the thermodynamic model.<br />
<br />
The functional groups in the block diagram are one of two fundamental frameworks used to describe an instrument. The other sub-divides the instrument into categories that often mirror historical areas of engineering. The former is focused on what will be done, including the overall scientific goals and the physical processes to be measured. The latter is focused on how it will be done, e.g. using optics, electronics or perhaps a practical knowledge of thermal management. A good engineer will constantly be thinking of the instrument in both frameworks. <br />
<br />
==Mechanical subsystem==<br />
<br />
[[Image:DNA_Setup_v2_Iso.png|600 px|thumb|center|DNA Melting System Model]]<br />
<br />
Mechanical support for the heating block, sample and optics will be built on a portable optical breadboard from [http://www.thorlabs.com ThorLabs]. Most of the components in the lab are from this supplier because the selection is large, the quality is quite good and the price reasonable. Although you are free to modify the design as the lab progresses, begin a build from the standard design shown above and demonstrated by the example in the lab. Gather the following Thorlabs components but do not start to assemble.<br />
<br />
* 1 MB1224 optical breadboard<br />
* 2 CL5 clamps (or similar)<br />
* 2 1/4-20 5/8" screws<br />
* 1 sheet metal support bracket<br />
* 4 1/4-20 1/4" screws<br />
<br />
==Thermal subsystem==<br />
The thermal subsystem includes all components to control heat flow into and out of the sample and monitor its temperature. Heating is accomplished using thermal electric coolers (TECs) in reverse. The hot side is placed in direct contact with the heating block, which is in direct contact with the sample vial. The TECs will be powered by manually controlling the Diablotek power supply. To avoid contamination of the sample or perturbation of its temperature, the temperature of a resistive temperature detector (RTD) embedded in the heating block will be monitored as a proxy for the sample temperature. In this part of the lab, air convection will cool down the sample when the Diablotek is switched off. The heat sink will help and the fan may be connected to a triple-output power supply to help, but these are included primarily for use in Part 2.<br />
<br />
===Parts===<br />
Gather the following components from the bins on the Center Cabinets, the DNA Melting drawer at the wet bench or on the East Cabinets:<br />
<br />
[[Image:CenterCabinets.jpg|right|250px|thumb|'''Center Cabinets''' The bins for most parts that you need will be set out on top of these cabinets, roughly in the center of the lab.]]<br />
[[Image:WetBench.jpg|right|250px|thumb|'''Wet Bench''' Supplies for our wet bench can be found in the three drawers at the left of the bench, the drawer left of the sink and on the wall behind and to the left as you face the wet bench. The drawer at the right of the wet bench contains various supplies for the DNA Melting instrument.]]<br />
[[Image:EastCabinets.jpg|right|250px|thumb|'''East Cabinets''' The center shelves of the East Cabinets contain the electronic breadboards, breadboard jumper wire, TEC connectors and TEC power supplies. Often these supplies are laid out on top of these cabinets.]]<br />
<br />
* A metal support bracket, having a fan, heatsink, 2 TEC heaters/coolers, and a heating block attached to it (if they're not attached, you'll need to attach them).<br />
* 3 wire nuts<br />
* 1 Diablotek 250 W power supply<br />
* 1 red/black TEC extension wire assembly<br />
<br />
===Assembly instructions===<br />
<br />
[[Image:TEC_picture.jpg|150 px|thumb|right|TEC showing hot side up when current flows into the red lead]]<br />
<br />
Ensure that the following have been done/attached to your metal bracket:<br />
# Attach the fan on the side of the support bracket using the 6-32 screws and nuts.<br />
# Attach the 2x2 heat sink to the bottom of the support bracket using two 4-40 screws at opposite corners.<br />
# Attach the support bracket to the MB1224 breadboard using 1/4-20 1/4" screws.<br />
# Now place the TECs on a napkin or two, with the red lead extending down and to the ''left'' as shown in the figure.<br />
# Apply a very thin layer of thermal grease to the exposed side of one of the TECs using a wooden stick from the drawer.<br />
#* Use a small amount of grease. Too much is worse than none.<br />
#* Best practice is to apply a baby-pea-sized amount, spread with the stick, smooth and remove excess almost to the point of total removal.<br />
# Now place the second TEC on top of the first.<br />
#* If the two TECs slide freely against each other, then there is too much grease.<br />
#** Separate the TECs and smooth/remove the grease until a modest force is required to slide them.<br />
#* Verify that the colors of the leads match. If not, flip one TEC.<br />
# Connect the black lead of the top TEC to the red lead of the bottom TEC using a wire nut.<br />
# Connect the TEC to the extension wire assembly using a wire nut to join the white wire to the red lead of the top TEC and the black wire to the black lead on the bottom TEC.<br />
# Join the white connector on the assembly to the black 2x2 connector of the Diabloteck.<br />
# Test the TECs by holding the them between thumb and finger while briefly flipping the switch on the Diablotek.<br />
#* One side should immediately heat up while the other immediately cools.<br />
#* The top side should be the hot side when the red wires extend down and to the '''left'''.<br />
#* If the Diablotek immediately shuts off (the fan will stop spinning) then there is a short. Check all connections.<br />
# Once the TEC assembly checks out, butter the bottom side of the assembly and place that side on the heat sink with the leads facing to the right.<br />
# Finally, apply thermal grease to the top side of the TEC assembly, place the heating block on top and anchor to the heat sink using the 1/2" long 4-40 screws and plastic washers.<br />
<br />
==Optical subsystem==<br />
<br />
A good optical subsystem design will be compact, stable and simple. It includes the optical components of the excitation and fluorescence detection functional groups from the block diagram. A good design will efficiently illuminate the sample and collect the maximum possible amount of emitted fluorescent light. There are a variety of ways to construct the apparatus. Use the model above and the example system in the lab as guides.<br />
<br />
The following is a rough set of instructions if you're not clear on how to replicate the example system in the lab. In addition to the following parts, you might want to add a few lenses to collimate your LED onto your sample, and to focus the light coming from your sample onto your photodiode. <br />
<br />
* 2 PH2 post holders<br />
* 2 BA1 post holder bases<br />
* 2 1/4-20 1/4" screws<br />
* 2 1/4-20 5/8" screws<br />
* 2 1/4" washers<br />
* 4 CP02 cage plates<br />
* 2 8-32 1/2" setscrews<br />
* 2 TR2 posts<br />
* 6 ER3 cage rails<br />
* 12 4-40 1/4" setscrews (or similar length)<br />
* 4 SML05 lens tubes<br />
* 4 SM1LTRR retaining rings<br />
* 1 blue LED PCB<br />
* 1 D470/40 excitation filter<br />
* 1 E515LPv2 emission filter<br />
* 1 SM1A6 adapter between SM1 and SM05 threads<br />
* 1 SM05PD1A mounted photodiode<br />
<br />
===Assembly instructions===<br />
<br />
# Attach each PH2 to a BA1 using 1/4" screws.<br />
# Attach two of the CP02 cage plates to two TR2 posts using an 8-32 setscrew.<br />
# Insert the TR2/CP02 assembly into the PH2/BA1 assemblies and lock in place with the TS25H setscrews.<br />
# Insert three ER4 cage rails into each CP02 above and lock in place with SS4S025 set screws or similar.<br />
#* Leave the blank hole on the bottom of the assembly.<br />
# Attach one of the remaining CP02 cage plates on the free end of each assembly above and lock in place.<br />
# Attach each of the above assemblies to the MB1224 using a washer and 1/4-20 screw less than 1" in length.<br />
# Arrange on the MB1224 and adjust CP02 heights as desired.<br />
# Mount the LED PCB in an SML05 lens tube and fix with a retaining ring.<br />
# Mount the excitation and emission filters in each of two lens tubes and fix with retaining ring.<br />
#* The arrow label on the thickness of the filter points ''toward the light source''.<br />
# Thread the SM1A6 adapter into a lens tube and fix with a retaining ring.<br />
# Thread the photodiode into the SM1A6 adapter.<br />
#* Teflon pipe thread tape can be used to hold the photodiode in the desired position.<br />
# Mount each lens tube in the appropriate CP02 cage plates.<br />
# Arrange each the resultant assemblies on the breadboard to most fully illuminate the sample vial and to gather the most of the resultant light on the photodiode.<br />
<br />
==Electronics subsystem==<br />
<br />
The electrical subsystem includes the LED with a current-limiting resistor, the photodiode, the transimpedance amplifier and a temperature detection circuit. The remainder of this section provides an example of each of these systems and instructions to build and test each one. Nevertheless, alternate ideas are welcome. Feel free to discuss with the Instructors.<br />
<br />
===Parts===<br />
<br />
Gather the following components from the electronics drawer to the right of the optics drawers, from the counter top opposite the wet bench and from the cable organizer on the East wall to the right of the white board.<br />
* 1 electronic breadboard<br />
* 1 jump wire kit<br />
* 2 red 3' banana cable or similar<br />
* 1 black 3' banana cable or similar<br />
* individual wire, as-needed from the scrap wire box or the wire spools opposite the wet bench<br />
* 1 62 &Omega; resistor from the center counter top on the South wall<br />
* 1 15 k&Omega; resistor<br />
* other resistors, as-needed for the amplifier<br />
* 1 blue LED from the DNA Melting drawer at the far right of the wet bench<br />
* 2 LF411 op-amps from the electronics drawer<br />
* 4 capacitors between 0.1 and 22 uF from either the electronics drawer or the counter top where the screws are stored.<br />
<br />
===Assembly instructions===<br />
<br />
====LED circuit====<br />
The led circuit consists of a 62 &Omega; resistor in series with a blue, jumbo LED. The circuit will be powered by the 5 volt fixed output of the lab power supply. The 62 &Omega; resistor prevents excessive current from flowing through the LED. Excessive current will let the smoke out of the LED.<br />
<br />
# Build the LED circuit on your electronic breadboard.<br />
#* Don't forget to connect the resistor. Directly connecting the LED to 5 V will blow out the LED.<br />
# Disable the output of the lab power supply.<br />
# Connect the LED circuit to the fixed 5 V output.<br />
<br />
====Temperature detection circuit====<br />
A platinum RTD is embedded inside the heating block. The resistance of the RTD varies with temperature according to the equation ''R<sub>RTD</sub>''=1000 &Omega; + 3.85 ''&theta;'', where ''&theta;'' is the temperature of the RTD in degrees Celsius. The easiest way to make a temperature measurement is to connect the RTD in series with a fixed resistor and a power supply. The output voltage of the resulting divider circuit varies as a function of temperature. <br />
<br />
The maximum current of the RTD is 1 mA. You will use a 15 V supply to the circuit, so the resistor must be at least 14 k&Omega;. Use a 15 k&Omega; resistor.<br />
<br />
# '''Find an equation for ''V<sub>out</sub>'' as a function of temperature. What voltage do you expect given an input voltage of 15 V with the RTD at room temperature.'''<br />
# Turn off the output of the triple-output power supply.<br />
# Connect the RTD to the electronic breadboard.<br />
# '''Measure the resistance of the 15 k&Omega; resistor with a DMM and record the value.'''<br />
# Connect the 15k&Omega; resistor in series with the RTD.<br />
# Set the power supply for '''SERIES''' operation.<br />
# Set the output voltage to 15 V on the supply and make note of its exact value using the DMM.<br />
# Set the current limit to 0.1 A.<br />
# Connect the positive terminal of '''CH1''' to a banana terminal on the breadboard for +15 V and wire it to the top red bus strip for +15 V supply.<br />
# Connect the negative terminal of '''CH2''' to a banana terminal on the breadboard for -15 V.<br />
# Connect the positive terminal of '''CH2''' to the black terminal on the breadboard for a ground reference and wire it to the top blue bus strip.<br />
#* Use three jump wires to minimize the parasitic voltage drop due to the breadboard tie points.<br />
# Connect the +15 V bus strip to the second vertical, red bus strip and wire this bus strip to the top of your 15 k&Omega; resistor.<br />
# Turn on the output of the power supply and measure the voltage across the RTD using the DMM.<br />
# '''What is the measured voltage of the RTD at room temperature and how does it compare to your predictions above?'''<br />
# '''What is the temperature corresponding to this measured voltage and how does it compare to some other measure of room temperature from a method of your choosing?'''<br />
# '''How is the temperature calculation affected by a 1% change in the 15 V input voltage and in the pull-up resistor, respectively?'''<br />
<br />
====Photodiode amplifier circuit====<br />
<br />
The current produced by the photodiode detector is a few tens of nanoamperes. This very small current must be amplified to a level where it can be easily measured. The data acquisition system you will use can measure voltages between + and -10 V. Use the two-stage op amp circuit shown below to convert the photodiode current to a voltage.<br />
<br />
<center>[[Image:Part1_TransimpedanceAmplifier.jpg|700 px|thumb|center|Multi-stage photodiode amplification circuit]]</center><br />
<br />
You will have to decide how to distribute the gain between the two op amp stages in the circuit. It is best to put as much gain in early stages of a multi-stage amplifier as possible. However, when the gain of a single stage becomes very high, nonidealities of the op amp become bothersome. The first stage of the amplifier will behave reasonably well as long as the gain is less than about 10<sup>6</sup> &Omega;.<br />
<br />
# '''Analyze the photodiode amplifier circuit. Choose resistor values to achieve an overall gain that will yield an output of about 5 V when the input is 500 nA.'''<br />
# Construct the circuit on your breadboard.<br />
#* Disable the power supply before you build.<br />
#* Do not yet connect the photodiode.<br />
#* Orient the LF411 op-amps so that pin 1, which is marked with a dot on the package, is at the upper left.<br />
# Connect the +/- 15 V bus strips to power the op-amps power pins.<br />
# Connect the vertical blue bus strips to the horizontal blue bus strip.<br />
<br />
=====Test connectivity and basic operation=====<br />
<br />
# Verify that all your component connections are correct and match the design.<br />
# Check that wires and components are properly seated in the breadboard and not suspiciously loose.<br />
#* You can use the ohm-meter function of the digital multi-meter to verify continuity.<br />
# Turn on the power supply and verify that +15V appears on pin 7 and -15V on pin 4.<br />
# Verify that pins 2 and 3 (the V- and V+ inputs to the op-amp) are equal.<br />
# '''Measure the output voltages of IC1 and IC2.'''<br />
#* Without the photodiode, the input current is zero but you may still see a significant offset voltage from 50 to 100 mV at IC1 pin 6.<br />
#* Refer to [http://measure.mit.edu/~20.309/wiki/index.php?title=Real_electronics#Real_op-amps Real Electronics, real op-amps] to understand, as well as to account for, this non-ideal op-amp behavior.<br />
<br />
==Data acquisition hardware subsystem==<br />
<br />
{{:20.309:DAQ System}}<br />
<br />
====Summary of DAQ inputs/outputs====<br />
<br />
A special connector has been prepared for you, connected as shown below. The connections and cable wire colors are also summarized [http://dl.dropbox.com/u/12957607/DAQ_Cable_Assembly_for_DNA_melting_lab.pdf in a memo].<br />
<br />
{| class="wikitable"<br />
|-<br />
! Signal Name<br />
! Signal Location<br />
! Ground Location<br />
! Pin wire color<br />
|-<br />
! DAQ Inputs <br />
|-<br />
! RTD<br />
| AI0+<br />
| AI0-<br />
| +Orange / -Black [lone pair, not with third (red) wire]<br />
|-<br />
! Photodiode <br />
| AI1+ <br />
| AI1-<br />
| +Green / -White<br />
|}<br />
<br />
<center>[[Image:DAQ_Wire_Colors.png|450 px|thumb|center|DAQ Connection Cable]]</center><br />
<br />
==DNAMelter software==<br />
<br />
[[Image:BasicDNAMelterIcon.png|75 px|left]]<br />
Use the <code>Basic DNA Melter GUI</code> program (located on the lab computer desktop) to collect data from the apparatus.<br />
<br />
At the end of an experimental run, use the "Save" button to write the date to a file. The data will be tab-delimited and can be read into Matlab with the <code>load</code> command.<br />
<br />
Documentation for the DNAMelter software is available here: [[DNA Melting: Using the Basic DNAMelter GUI]]<br />
<br />
====If you need to debug the DAQ (skip otherwise!)====<br />
* When debugging problems, it is frequently useful to ascertain whether the data acquisition system is working properly. National Instruments provides an application for this purpose called Measurement and Automation Explorer. Use Measurement and Automation Explorer to verify that the DAQ is properly connected to and communicating with the computer. The program also provides the ability to manually control all of the DAQ system's inputs and outputs.<br />
** Launch Measurement and Automation Explorer by selecting Start->All Applications->National Instruments->Measurement and Automation.<br />
** In the "Configuration" tab, select "Devices and Interfaces" and then "NI-DAQMx Devices." Select "Dev1."<br />
** If Dev 1 does not show up, the problem is that computer cannot communicate with the DAQ system. Ensure that the USB cable is connected to the DAQ system. If the cable is connected and the device still does not show up, unplug the USB cable, count to five, and plug it back in. <br />
** If the DAQ shows up under a different name, disconnect the USB cable. Delete all the "NI USB-6xxx" entries and then reconnect the USB cable. Verify that one entry reappears and that it is called "Dev1." <br />
** Select the "Test Panels" tab to manually control or read signals from the DAQ.<br />
* If you run DNAMelter and there is a data acquisition error, for example, "<code>Data acquisition cannot start!</code>", open Measurement and Automation Explorer and follow the steps above. Run DNAMelter again. If it still doesn't work, grab a beer or ask an instructor for help.<br />
* The DAQ has only one analog-to-digital converter (ADC). A multiplexer circuit sequentially samples voltages from the analog inputs configured in the control software. If any of the inputs is outside the allowable range of +/-10V, all of the inputs may behave erratically. The voltages reported may look reasonable so this can be very misleading. If none of the DAQ readings make sense, ensure that all of the analog voltages are in the allowable range and reset the DAQ by cycling the power of the DAQ (use the switch on the 6341 or disconnect the USB of the 6212).<br />
* If the DAQ gets short-circuited, it may crash. If this happens, Matlab will frequently give a deceptive error message, for example claiming that the limits on the band-pass filter do not satisfy some condition. In this case, correct the connection and cycle the DAQ power.<br />
<br />
==Part 1 measurements==<br />
<br />
Please read this entire section before beginning. Then start at Testing the instrument, with reference back to the sample sections as-needed.<br />
<br />
===Making samples===<br />
<br />
{{Template:Biohazard warning|message=LC Green in DMSO is readily absorbed through skin. Synthetic oligonucleotides may be harmful by inhalation, ingestion, or skin absorption. Wear gloves when handling samples. Wear safety goggles at all times when pipetting the LC Green/DNA samples. Do not create aerosols. The health effects of LC Green have not been thoroughly investigated. See the LC Green and synthetic oligonucleotide under <code>../EHS Guidelines/MSDS Repository</code> in the course locker for more information.}}<br />
<br />
====Sample prep steps====<br />
<br />
#Pipet 500 &mu;L of DNA plus dye solution into a glass vial. <br />
#Pipet up to 200 &mu;L of mineral oil on top of the sample to help prevent evaporation. It will help to pipette into the corners first. Be careful not to get oil on the cuvette sides far above the sample. It will run down during your experiment and cause shifts in the photodiode signal. Place the cap loosely on the vial. <br />
#*Keep the sample vertical to make sure the oil stays on top. <br />
<br />
* You can use the same sample for several heating/cooling cycles.<br />
* Only discard a sample if you lose significant volume due to evaporation or if your signal gets too low. <br />
<br />
To clean your glass vial between samples, flush with alcohol in the waste container and rinse with water at the drain. Suck out residual liquid with the vacuum and drawn Pasteur pipette to the left of the sink.<br />
<br />
====Sample disposal====<br />
<br />
{{Template:Environmental Warning|message=Discard pipette tips with DNA sample residue in the pipette tips or the ''Biohazard Sharps'' container. Do not pour synthetic oligonucleotides with LC Green down the drain. Pour your used samples into the waste container provided in the middle of the wet bench.}}<br />
<br />
===Testing the instrument===<br />
<br />
Once your apparatus is built, use samples of fluorescein (~ 30&mu;M concentration) as a stand-in for LC Green + DNA to test the instrument. Fluorescein is cheap, non-toxic and will not bleach as quickly as LC Green.<br />
<br />
====Making a rough measurement of the SNR====<br />
<br />
Carry out a simple estimate of the signal to noise ratio (SNR) of the instrument by comparing the difference between the average signal with a sample present and with water as a null reference. Calculate the noise level with no sample present. This process requires 500 uL of 30 uM fluorescein and a vial containing the same amount of water. <br />
<br />
# Use <code>Basic DNA Melter GUI</code> to record the photodiode signal with the water vial in the heating block. <br />
# Start by clearing the data and then wait about 10 s. <br />
# Replace the water vial with the DNA of fluorescein vial and record for another 10 s then save the data. <br />
#* Be sure that all other conditions of the instrument are stable throughout the test. <br />
# Plot this data in Matlab and and compare the average signal levels for the water segment of the data and the fluorescein segment.<br />
#* The difference between the two average signal levels is the "signal" portion of the signal-to-noise ratio (SNR) calculation. <br />
# Now measure the noise. Subtract the average value from the fluorescein portion of the signal. This is the noise in your measurement. Measure the standard deviation of this residual. This is your initial estimate of the noise level.<br />
# Add any estimate of the long-term noise to the Std Dev calculated from the signal. This is the "noise" portion of the SNR.<br />
# '''Calculate the SNR as signal divided by noise.'''<br />
<br />
===Experiment steps===<br />
<br />
Once the instrument is confirmed to be functioning properly, follow these instructions to generate melting curve data for the 20 bp DNA-LC Green solution.<br />
<br />
#Open and run the <code>Basic DNA Melter GUI</code> in Matlab and follow the instructions there.<br />
# Confirm that block temperature is near room temperature when the Diablotek supply is off.<br />
# Apply heat to the block by switching the Diablotek on to increase the block temperature to 95&deg;C, toggle the Diablotek power in order to hold the temperature long enough for the fluorescence curve to reach a minimum, then allow the block to cool down by switching off the Diablotek.<br />
#* Other approaches may be used. One goal would be to heat the block more slowly, possibly by toggling the current while heating.<br />
# Now prepare a sample using the stock of "beater" DNA.<br />
# Place the vial in the instrument, but keep the LED off until ready to measure the melting curve.<br />
# Measure melting curves of the beater DNA sample as-desired to understand instrument operation.<br />
# Prepare a sample using fresh 20 bp 100 mM KCl DNA.<br />
# Measure a melting curve of the fresh sample.<br />
#* Be sure to save a copy in a local directory.<br />
<br />
{{:DNA Melting Report Requirements for Part 1}}<br />
<br />
==Lab manual sections==<br />
<br />
*[[Lab Manual:Measuring DNA Melting Curves]]<br />
*[[DNA Melting: Simulating DNA Melting - Basics]]<br />
*[[DNA Melting Part 1: Measuring Temperature and Fluorescence]]<br />
*[[DNA Melting Report Requirements for Part 1]]<br />
*[[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]<br />
*[[DNA Melting Report Requirements for Part 2]]<br />
<br />
==References==<br />
<references/><br />
<br />
==Subset of datasheets==<br />
(Many more can be found online or on the course share)<br />
#[http://www.ni.com/pdf/manuals/371931f.pdf National Instruments USB-6212 user manual]<br />
#[http://www.ni.com/pdf/manuals/370784d.pdf National Instruments USB-6341 user manual]<br />
#[http://www.national.com/ds/LF/LF411.pdf Op-amp datasheet]<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/Manta_G032_camera_measurementsManta G032 camera measurements2015-10-22T00:26:40Z<p>Steven Nagle: /* Calculations */</p>
<hr />
<div>{{Template:20.309}}<br />
<br />
==Overview==<br />
This page contains data from the demo I did in lecture on 9/22/2015 of the Manta. The point of the demo was to measure the gain <math>g</math>, dark current <math>i_d</math>, and read noise <math>N_r</math> of the Manta G032 cameras we use in the microscopy lab. Note that I showed a linear plot in lecture and the plot below is log-log.<br />
<br />
* Gain relates the binary value reported by the camera, referred to here as ''counts'', to the number of electrons collected in a pixel: <math>P_{x,y}=g N_{x,y}</math>, where <math>P_{x,y}</math> is the value reported by the camera at pixel location <math>x</math>, <math>y</math>, and <math>N_{x,y}</math> is the number of electrons detected.<br />
* Dark current is the average number of dark electrons that are collected in units of electrons per second.<br />
* Read noise is a roughly Gaussian distributed random variable that lumps together noise sources that arise when counting electrons.<br />
<br />
==Measurement procedure==<br />
[[File:Manta Noise Measurement.png|right|thumb|400 px]]<br />
* Direct a light source at the camera to produce a range of intensities on the surface of the detector.<br />
* Record a 100 frame movie of the light source at 20 FPS with an exposure of 150 &mu;s.<br />
* Turn off the light source and record a 100 frame dark movie with identical exposure settings.<br />
* Compute a single, average dark image by averaging all frames of the dark movie.<br />
* Subtract the dark image from each frame of the light movie.<br />
* Compute the variance of each pixel (noise squared) and plot versus the average value (signal).<br />
<br />
==Calculations==<br />
The value of a particular pixel over a certain time interval, <math>P_{x,y}[t]</math>, is equal to the sum of the number of photoelectrons and the number of dark electrons counted during that interval plus the number of electrons gained or lost due to read noise, times the gain <math>g</math>. (The square brackets indicate that <math>P_{x,y}</math> is evaluated at discrete time points.) Mathematically:<br />
<br />
:<math>P_{x,y}[t]=g \left(I_{x,y}[t]+R_{x,y}[t]+D_{x,y}(t)) \right)</math>,<br />
where<br />
* <math>I_{x,y}[t]</math> is the number of photoelectrons generated during interval <math>t</math>,<br />
* <math>R_{x,y}[t]</math> is the read noise during time interval <math>t</math>,<br />
* and <math>D_{x,y}[t]</math> is the number of dark current electrons generated during time interval <math>t</math>.<br />
<br />
The next step is to write an expression for the mean value of each pixel. Means of terms in a sum add, so <math>\langle (P_{x,y}) \rangle</math> can be found by summing the means of the three individual terms. The mean value of read noise is zero, and the mean value of dark current is <math>i_d \delta t</math>, which gives:<br />
<br />
:<math>\langle P_{x,y}\rangle = g\left(\langle I_{x,y}\rangle + i_d \delta t \right)</math><br />
<br />
We subtracted the average dark frame to remove <math>i_d \delta t</math>, so the value plotted on the horizontal axis is just <math>g\left(\langle I_{x,y}\rangle \right)</math>. Cool.<br />
<br />
Now we need an expression for the noise. Variances of a sum of terms also add, so <math>\operatorname{Var}(P_{x,y})</math> can be found by summing the variances of the three individual terms. The photoelectron count, <math>I_{x,y}</math>, is Poisson distributed, so its variance is equal to its mean: <math>\operatorname{Var}(I_{x,y})=\langle I_{x,y} \rangle</math>. The second term has a constant variance that is a property of the camera, the read noise <math>N_r</math>. The third term is also Poisson distributed, with an average value of <math>i_d \delta t</math>, where <math>\delta t</math> is the exposure time. This gives:<br />
<br />
:<math>\text{Var}\left(P_{x,y}\right)=g\left(\langle I_{x,y}\rangle+N_r^2 + i_d \delta t \right)</math><br />
<br />
The x-intercept is equal to read noise plus dark noise times <math>g</math>. The slope of the line is equal to <math>g</math>.<br />
<br />
==Results==<br />
The slope of the line, <math>g</math>, is equal to 0.2987, meaning that the camera produces approximately 1 count per 3.3 electrons.<br />
<br />
The intercept is around 15 counts, meaning that dark plus read noise is about <math>\sqrt{15}=3.9</math> counts, or about 13 electrons at this (short) exposure time.<br />
<br />
I took another dataset with a 1 second exposure so we can to figure out the relative contributions of dark and read noise. I will post soon.<br />
<br />
For comparison, the [http://www.hamamatsu.com/eu/en/community/life_science_camera/product/search/C11440-22CU/index.html Orca Flash 4.0 scientific CMOS camera] has a read noise of 1.6 electrons and a dark current of 0.06 electrons per second (with air cooling). The Flash 4.0 cools its detector to achieve such a low dark current. The camera also provides a slow-readout mode in which the read noise is reduced to 1.4 electrons. The gain of the Flash 4.0 is 2.<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/Manta_G032_camera_measurementsManta G032 camera measurements2015-10-22T00:23:14Z<p>Steven Nagle: /* Overview */</p>
<hr />
<div>{{Template:20.309}}<br />
<br />
==Overview==<br />
This page contains data from the demo I did in lecture on 9/22/2015 of the Manta. The point of the demo was to measure the gain <math>g</math>, dark current <math>i_d</math>, and read noise <math>N_r</math> of the Manta G032 cameras we use in the microscopy lab. Note that I showed a linear plot in lecture and the plot below is log-log.<br />
<br />
* Gain relates the binary value reported by the camera, referred to here as ''counts'', to the number of electrons collected in a pixel: <math>P_{x,y}=g N_{x,y}</math>, where <math>P_{x,y}</math> is the value reported by the camera at pixel location <math>x</math>, <math>y</math>, and <math>N_{x,y}</math> is the number of electrons detected.<br />
* Dark current is the average number of dark electrons that are collected in units of electrons per second.<br />
* Read noise is a roughly Gaussian distributed random variable that lumps together noise sources that arise when counting electrons.<br />
<br />
==Measurement procedure==<br />
[[File:Manta Noise Measurement.png|right|thumb|400 px]]<br />
* Direct a light source at the camera to produce a range of intensities on the surface of the detector.<br />
* Record a 100 frame movie of the light source at 20 FPS with an exposure of 150 &mu;s.<br />
* Turn off the light source and record a 100 frame dark movie with identical exposure settings.<br />
* Compute a single, average dark image by averaging all frames of the dark movie.<br />
* Subtract the dark image from each frame of the light movie.<br />
* Compute the variance of each pixel (noise squared) and plot versus the average value (signal).<br />
<br />
==Calculations==<br />
The value, counts reported by the camera, of a particular pixel over a certain time interval, <math>P_{x,y}[t]</math>, is equal to the sum of the number of photoelectrons and the number of dark electrons counted during that interval plus the number of electrons gained or lost due to read noise, times the gain, <math>g</math>. (The square brackets indicate that <math>P_{x,y}</math> is evaluated at discrete time points.) Mathematically:<br />
<br />
:<math>P_{x,y}[t]=g \left(I_{x,y}[t]+R_{x,y}[t]+D_{x,y}(t)) \right)</math>,<br />
where<br />
* <math>I_{x,y}[t]</math> is the number of photoelectrons generated during interval <math>t</math>,<br />
* <math>R_{x,y}[t]</math> is the read noise during time interval <math>t</math>,<br />
* and <math>D_{x,y}[t]</math> is the number of dark current electrons generated during time interval <math>t</math>.<br />
<br />
The next step is to write an expression for the mean value of each pixel. Means of terms in a sum add, so <math>\langle (P_{x,y}) \rangle</math> can be found by summing the means of the three individual terms. The mean value of read noise is zero, and the mean value of dark current is <math>i_d \delta t</math>, which gives:<br />
<br />
:<math>\langle P_{x,y}\rangle = g\left(\langle I_{x,y}\rangle + i_d \delta t \right)</math><br />
<br />
We subtracted the average dark frame to remove <math>i_d \delta t</math>, so the value plotted on the horizontal axis is just <math>g\left(\langle I_{x,y}\rangle \right)</math>. Cool.<br />
<br />
Now we need an expression for the noise. Variances of a sum of terms also add, so <math>\operatorname{Var}(P_{x,y})</math> can be found by summing the variances of the three individual terms. The photoelectron count, <math>I_{x,y}</math>, is Poisson distributed, so its variance is equal to its mean: <math>\operatorname{Var}(I_{x,y})=\langle I_{x,y} \rangle</math>. The second term has a constant variance that is a property of the camera, the read noise <math>N_r</math>. The third term is also Poisson distributed, with an average value of <math>i_d \delta t</math>, where <math>\delta t</math> is the exposure time. This gives:<br />
<br />
:<math>\text{Var}\left(P_{x,y}\right)=g\left(\langle I_{x,y}\rangle+N_r^2 + i_d \delta t \right)</math><br />
<br />
The x-intercept is equal to read noise plus dark noise times <math>g</math>. The slope of the line is equal to <math>g</math>.<br />
<br />
==Results==<br />
The slope of the line, <math>g</math>, is equal to 0.2987, meaning that the camera produces approximately 1 count per 3.3 electrons.<br />
<br />
The intercept is around 15 counts, meaning that dark plus read noise is about <math>\sqrt{15}=3.9</math> counts, or about 13 electrons at this (short) exposure time.<br />
<br />
I took another dataset with a 1 second exposure so we can to figure out the relative contributions of dark and read noise. I will post soon.<br />
<br />
For comparison, the [http://www.hamamatsu.com/eu/en/community/life_science_camera/product/search/C11440-22CU/index.html Orca Flash 4.0 scientific CMOS camera] has a read noise of 1.6 electrons and a dark current of 0.06 electrons per second (with air cooling). The Flash 4.0 cools its detector to achieve such a low dark current. The camera also provides a slow-readout mode in which the read noise is reduced to 1.4 electrons. The gain of the Flash 4.0 is 2.<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/Manta_G032_camera_measurementsManta G032 camera measurements2015-10-21T23:58:24Z<p>Steven Nagle: /* Results */</p>
<hr />
<div>{{Template:20.309}}<br />
<br />
==Overview==<br />
This page contains data from the demo I did in lecture on 9/22/2015 of the Manta. The point of the demo was to measure the gain <math>g</math>, dark current <math>i_d</math>, and read noise <math>N_r</math> of the Manta G032 cameras we use in the microscopy lab. Note that I showed a linear plot in lecture and the plot below is log-log.<br />
<br />
* Gain relates the binary value reported by the camera to the number of electrons collected in a pixel: <math>P_{x,y}=g N_{x,y}</math>, where <math>P_{x,y}</math> is the value reported by the camera at pixel location <math>x</math>, <math>y</math>, and <math>N_{x,y}</math> is the number of electrons detected.<br />
* Dark current is the average number of dark electrons that are collected in units of electrons per second.<br />
* Read noise is a roughly Gaussian distributed random variable that lumps together noise sources that arise when counting electrons.<br />
<br />
==Measurement procedure==<br />
[[File:Manta Noise Measurement.png|right|thumb|400 px]]<br />
* Direct a light source at the camera to produce a range of intensities on the surface of the detector.<br />
* Record a 100 frame movie of the light source at 20 FPS with an exposure of 150 &mu;s.<br />
* Turn off the light source and record a 100 frame dark movie with identical exposure settings.<br />
* Compute a single, average dark image by averaging all frames of the dark movie.<br />
* Subtract the dark image from each frame of the light movie.<br />
* Compute the variance of each pixel (noise squared) and plot versus the average value (signal).<br />
<br />
==Calculations==<br />
The value, counts reported by the camera, of a particular pixel over a certain time interval, <math>P_{x,y}[t]</math>, is equal to the sum of the number of photoelectrons and the number of dark electrons counted during that interval plus the number of electrons gained or lost due to read noise, times the gain, <math>g</math>. (The square brackets indicate that <math>P_{x,y}</math> is evaluated at discrete time points.) Mathematically:<br />
<br />
:<math>P_{x,y}[t]=g \left(I_{x,y}[t]+R_{x,y}[t]+D_{x,y}(t)) \right)</math>,<br />
where<br />
* <math>I_{x,y}[t]</math> is the number of photoelectrons generated during interval <math>t</math>,<br />
* <math>R_{x,y}[t]</math> is the read noise during time interval <math>t</math>,<br />
* and <math>D_{x,y}[t]</math> is the number of dark current electrons generated during time interval <math>t</math>.<br />
<br />
The next step is to write an expression for the mean value of each pixel. Means of terms in a sum add, so <math>\langle (P_{x,y}) \rangle</math> can be found by summing the means of the three individual terms. The mean value of read noise is zero, and the mean value of dark current is <math>i_d \delta t</math>, which gives:<br />
<br />
:<math>\langle P_{x,y}\rangle = g\left(\langle I_{x,y}\rangle + i_d \delta t \right)</math><br />
<br />
We subtracted the average dark frame to remove <math>i_d \delta t</math>, so the value plotted on the horizontal axis is just <math>g\left(\langle I_{x,y}\rangle \right)</math>. Cool.<br />
<br />
Now we need an expression for the noise. Variances of a sum of terms also add, so <math>\operatorname{Var}(P_{x,y})</math> can be found by summing the variances of the three individual terms. The photoelectron count, <math>I_{x,y}</math>, is Poisson distributed, so its variance is equal to its mean: <math>\operatorname{Var}(I_{x,y})=\langle I_{x,y} \rangle</math>. The second term has a constant variance that is a property of the camera, the read noise <math>N_r</math>. The third term is also Poisson distributed, with an average value of <math>i_d \delta t</math>, where <math>\delta t</math> is the exposure time. This gives:<br />
<br />
:<math>\text{Var}\left(P_{x,y}\right)=g\left(\langle I_{x,y}\rangle+N_r^2 + i_d \delta t \right)</math><br />
<br />
The x-intercept is equal to read noise plus dark noise times <math>g</math>. The slope of the line is equal to <math>g</math>.<br />
<br />
==Results==<br />
The slope of the line, <math>g</math>, is equal to 0.2987, meaning that the camera produces approximately 1 count per 3.3 electrons.<br />
<br />
The intercept is around 15 counts, meaning that dark plus read noise is about <math>\sqrt{15}=3.9</math> counts, or about 13 electrons at this (short) exposure time.<br />
<br />
I took another dataset with a 1 second exposure so we can to figure out the relative contributions of dark and read noise. I will post soon.<br />
<br />
For comparison, the [http://www.hamamatsu.com/eu/en/community/life_science_camera/product/search/C11440-22CU/index.html Orca Flash 4.0 scientific CMOS camera] has a read noise of 1.6 electrons and a dark current of 0.06 electrons per second (with air cooling). The Flash 4.0 cools its detector to achieve such a low dark current. The camera also provides a slow-readout mode in which the read noise is reduced to 1.4 electrons. The gain of the Flash 4.0 is 2.<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/Manta_G032_camera_measurementsManta G032 camera measurements2015-10-21T23:48:15Z<p>Steven Nagle: /* Calculations */</p>
<hr />
<div>{{Template:20.309}}<br />
<br />
==Overview==<br />
This page contains data from the demo I did in lecture on 9/22/2015 of the Manta. The point of the demo was to measure the gain <math>g</math>, dark current <math>i_d</math>, and read noise <math>N_r</math> of the Manta G032 cameras we use in the microscopy lab. Note that I showed a linear plot in lecture and the plot below is log-log.<br />
<br />
* Gain relates the binary value reported by the camera to the number of electrons collected in a pixel: <math>P_{x,y}=g N_{x,y}</math>, where <math>P_{x,y}</math> is the value reported by the camera at pixel location <math>x</math>, <math>y</math>, and <math>N_{x,y}</math> is the number of electrons detected.<br />
* Dark current is the average number of dark electrons that are collected in units of electrons per second.<br />
* Read noise is a roughly Gaussian distributed random variable that lumps together noise sources that arise when counting electrons.<br />
<br />
==Measurement procedure==<br />
[[File:Manta Noise Measurement.png|right|thumb|400 px]]<br />
* Direct a light source at the camera to produce a range of intensities on the surface of the detector.<br />
* Record a 100 frame movie of the light source at 20 FPS with an exposure of 150 &mu;s.<br />
* Turn off the light source and record a 100 frame dark movie with identical exposure settings.<br />
* Compute a single, average dark image by averaging all frames of the dark movie.<br />
* Subtract the dark image from each frame of the light movie.<br />
* Compute the variance of each pixel (noise squared) and plot versus the average value (signal).<br />
<br />
==Calculations==<br />
The value, counts reported by the camera, of a particular pixel over a certain time interval, <math>P_{x,y}[t]</math>, is equal to the sum of the number of photoelectrons and the number of dark electrons counted during that interval plus the number of electrons gained or lost due to read noise, times the gain, <math>g</math>. (The square brackets indicate that <math>P_{x,y}</math> is evaluated at discrete time points.) Mathematically:<br />
<br />
:<math>P_{x,y}[t]=g \left(I_{x,y}[t]+R_{x,y}[t]+D_{x,y}(t)) \right)</math>,<br />
where<br />
* <math>I_{x,y}[t]</math> is the number of photoelectrons generated during interval <math>t</math>,<br />
* <math>R_{x,y}[t]</math> is the read noise during time interval <math>t</math>,<br />
* and <math>D_{x,y}[t]</math> is the number of dark current electrons generated during time interval <math>t</math>.<br />
<br />
The next step is to write an expression for the mean value of each pixel. Means of terms in a sum add, so <math>\langle (P_{x,y}) \rangle</math> can be found by summing the means of the three individual terms. The mean value of read noise is zero, and the mean value of dark current is <math>i_d \delta t</math>, which gives:<br />
<br />
:<math>\langle P_{x,y}\rangle = g\left(\langle I_{x,y}\rangle + i_d \delta t \right)</math><br />
<br />
We subtracted the average dark frame to remove <math>i_d \delta t</math>, so the value plotted on the horizontal axis is just <math>g\left(\langle I_{x,y}\rangle \right)</math>. Cool.<br />
<br />
Now we need an expression for the noise. Variances of a sum of terms also add, so <math>\operatorname{Var}(P_{x,y})</math> can be found by summing the variances of the three individual terms. The photoelectron count, <math>I_{x,y}</math>, is Poisson distributed, so its variance is equal to its mean: <math>\operatorname{Var}(I_{x,y})=\langle I_{x,y} \rangle</math>. The second term has a constant variance that is a property of the camera, the read noise <math>N_r</math>. The third term is also Poisson distributed, with an average value of <math>i_d \delta t</math>, where <math>\delta t</math> is the exposure time. This gives:<br />
<br />
:<math>\text{Var}\left(P_{x,y}\right)=g\left(\langle I_{x,y}\rangle+N_r^2 + i_d \delta t \right)</math><br />
<br />
The x-intercept is equal to read noise plus dark noise times <math>g</math>. The slope of the line is equal to <math>g</math>.<br />
<br />
==Results==<br />
The slope of the line = 0.2987, meaning that the camera produces approximately 1 count per 3.3 electrons.<br />
<br />
The intercept is around 15 counts, meaning that dark plus read noise is about <math>\sqrt{15}=3.9</math> counts, or about 13 electrons at this (short) exposure time.<br />
<br />
I took another dataset with a 1 second exposure so we can to figure out the relative contributions of dark and read noise. I will post soon.<br />
<br />
For comparison, the [http://www.hamamatsu.com/eu/en/community/life_science_camera/product/search/C11440-22CU/index.html Orca Flash 4.0 scientific CMOS camera] has a read noise of 1.6 electrons and a dark current of 0.06 electrons per second (with air cooling). The Flash 4.0 cools its detector to achieve such a low dark current. The camera also provides a slow-readout mode in which the read noise is reduced to 1.4 electrons. The gain of the Flash 4.0 is 2.<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/Optical_Microscopy:_Part_3_Report_OutlineOptical Microscopy: Part 3 Report Outline2015-10-20T22:02:24Z<p>Steven Nagle: </p>
<hr />
<div><br />
<ol start="6"><br />
<br />
</li> <li>Resolution<br />
<ol><br />
<li>Procedure<br />
<ul><li>Document the samples you used and how you captured images (camera settings, software used, etc…)</li></ul><br />
</li><br />
<li>Data</li><br />
<ul><br />
<li>Include an image of the PSF sample indicating which beads were used for resolution measurement..</li><br />
</ul><br />
<li>Analysis and Results</li><br />
<ul><br />
<li>Report the resolution you measured. Make sure to include N and a measure of uncertainty.</li><br />
<li>Show sample Gaussian fits.</li><br />
<li>Explain the Matlab algorithm used for data analysis.</li><br />
</ul><br />
<li>Discussion</li><br />
<ul><br />
<li>Compare the measured value to the theoretical value.</li><br />
<li>Include a thorough discussion of error sources. Do not comment on insignificant sources of error. To determine which error sources are significant, and which are not, you must think carefully about the uncertainty related to each error source and estimate its magnitude and sign. Include these estimates in your report along with your estimate of the combined, total uncertainty.</li><br />
</ul><br />
</ol><br />
</li><br />
</li> <br />
<li>Stability<br />
<ol><br />
<li>Procedure<br />
<ul><li>Document the samples you used and how you captured images (camera settings, frame rate, total number of frames, exposure, software used, etc…)</li></ul><br />
</li><br />
<li>Data</li><br />
<ul><br />
<li>Show an example frame from the stability movie.</li><br />
<li>Plot two or more example bead trajectories for each of the samples. (Hint: If you subtract the initial position from each trajectory, then you can plot multiple trajectories on a single set of axes.)<br />
</ul><br />
<li>Analysis and Results</li><br />
<ul><br />
<li>Plot MSD versus time interval &tau; for individual and difference tracks using log-log axes.</li><br />
<li>Provide a bullet point outline of data analysis methodology.</li><br />
<li>Include a thorough discussion of error sources.</li><br />
</ul><br />
<li>Discussion</li><br />
<ul><br />
<li>What are the benefits and drawbacks of differential tracking?</li><br />
</ul><br />
</ol><br />
</li><br />
</ol></div>Steven Naglehttp://measurebiology.org/wiki/Optical_Microscopy:_Part_3_Report_OutlineOptical Microscopy: Part 3 Report Outline2015-10-20T21:58:59Z<p>Steven Nagle: </p>
<hr />
<div><br />
<ol start="6"><br />
<br />
</li> <li>Resolution<br />
<ol><br />
<li>Procedure<br />
<ul><li>Document the samples you used and how you captured images (camera settings, software used, etc…)</li></ul><br />
</li><br />
<li>Data</li><br />
<ul><br />
<li>Include an image of the PSF sample indicating which beads were used for resolution measurement..</li><br />
</ul><br />
<li>Analysis and Results</li><br />
<ul><br />
<li>Report the resolution you measured. Make sure to include N and a measure of uncertainty.</li><br />
<li>Show sample Gaussian fits.</li><br />
<li>Explain the Matlab algorithm used for data analysis.</li><br />
</ul><br />
<li>Discussion</li><br />
<ul><br />
<li>Compare the measured value to the theoretical value.</li><br />
<li>Include a thorough discussion of error sources. Do not comment on insignificant sources of error. To determine which error sources are significant, and which are not, you must think carefully about the uncertainty related to each error source and estimate its magnitude and sign. Include these estimates in your report along with your estimate of the combined, total uncertainty.</li><br />
</ul><br />
</ol><br />
</li><br />
</li> <br />
<li>Stability<br />
<ol><br />
<li>Procedure<br />
<ul><li>Document the samples you used and how you captured images (camera settings, frame rate, total number of frames, exposure, software used, etc…)</li></ul><br />
</li><br />
<li>Data</li><br />
<ul><br />
<li>Show an example frame from the stability movie.</li><br />
<li>Plot two or more example bead trajectories for each of the samples. (Hint: If you subtract the initial position from each trajectory, then you can plot multiple trajectories on a single set of axes.)<br />
</ul><br />
<li>Analysis and Results</li><br />
<ul><br />
<li>Plot MSD versus time interval &tau; for individual and difference tracks. Use a linear or logarithmic vertical axis so as to most clearly illustrate the relationship between the two datasets.</li><br />
<li>Provide a bullet point outline of data analysis methodology.</li><br />
<li>Include a thorough discussion of error sources.</li><br />
</ul><br />
<li>Discussion</li><br />
<ul><br />
<li>What are the benefits and drawbacks of differential tracking?</li><br />
</ul><br />
</ol><br />
</li><br />
</ol></div>Steven Naglehttp://measurebiology.org/wiki/Optical_Microscopy:_Part_4_Report_OutlineOptical Microscopy: Part 4 Report Outline2015-10-20T21:51:53Z<p>Steven Nagle: </p>
<hr />
<div><ol start="5"><br />
</li> <li>Viscosity <br />
<ol><br />
<li>Procedure<br />
<ul><li>Document the samples you prepared and used and how you captured images (camera settings including frame acquisition rate, number of frames, number of particles in the region of interest, choice of sample plane, etc)</li></ul><br />
</li><br />
<li>Data</li><br />
<ul><br />
<li>Include a snapshot of the 0.84 &mu;m fluorescent beads monitored.</li><br />
<li>Plot two or more example bead trajectories for each of the samples. (Hint: If you subtract the initial position from each trajectory, then you can plot multiple trajectories on a single set of axes.)</li><br />
</ul><br />
<li>Analysis and Results</li><br />
<ul><br />
<li>Plot the average MSD vs τ results for all glycerin samples (A, B, C, and D); use log-log axes. Use the minimum number of axes that can convey your results clearly.</li><br />
<li>Include a table of the diffusion coefficient, viscosity and glycerin/water ratio for each of the samples (A, B, C, and D).</li><br />
<li>Plot the average MSD for all three PVP samples on a single set of log-log axes (0h, 1h, and 3h).</li><br />
<li>Provide a bullet point outline of all calculations and data processing steps.</li><br />
</ul><br />
<li>Discussion</li><br />
<ul><br />
<li>How do your viscosity calculations compare to your expectations? (This [https://dl.dropboxusercontent.com/u/12957607/Viscosity%20of%20Aqueous%20Glycerine%20Solutions.pdf chart] is a useful reference.)</li><br />
<li>What can you infer about the viscoelastic properties of the PVP gels from your MSD plots?</li><br />
<li>Include a thorough discussion of error sources and the approaches to minimize them.</li><br />
</ul><br />
</ol><br />
</li><br />
</li> <br />
</ol></div>Steven Naglehttp://measurebiology.org/wiki/Shot_noise_and_centroid_findingShot noise and centroid finding2015-10-15T13:59:05Z<p>Steven Nagle: </p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:Optical Microscopy Lab]]<br />
{{Template:20.309}}<br />
<br />
[[Image:Simulated Shot Noise.png|Simulated image of a fluorescent microshpere at various signal to noise ratios.]]<br />
<br />
Shot noise is a fluctuation that affects all light intensity measurements, including microscopic images recorded with a CCD camera. <ref>There are a few exotic methods, such as [http://www.rp-photonics.com/amplitude_squeezed_light.html amplitude-squeezed light] that reduce noise below the shot noise level.</ref> It is a consequence of the discrete and stochastic nature of photon emission. Poisson statistics provide an excellent model for shot noise. The standard deviation of a Poisson-distributed random variable is equal to the square root of its average value. Thus, the lowest possible signal to noise ratio of an intensity measurement is equal to the square root of the average number of photons (intensity). The simulated images above show the effect of shot noise on the image of a fluorescent microsphere with a radius of 10 pixels for several values of intensity. <br />
<br />
One method to estimate the position of a microsphere in an image is to compute an intensity-weighted centroid (similar to a center of mass calculation). Intensity-weighted centroids can provide locations accurate to a fraction of a pixel. Shot noise causes random variation in the pixel intensities, which affects the computed centroid. Even for a stationary particle, repeated centroid computations will exhibit random variation. Deriving an analytical expression for the variation is tedious. Happily, patient people with exceptional mathematical abilities have taken the time to do so. (See, for example, this reference: [http://www.opticsinfobase.org/josaa/abstract.cfm?uri=josaa-27-9-2038|Hui Jia, Jiankun Yang, and Xiujian Li. Minimum variance unbiased subpixel centroid estimation of point image limited by photon shot noise.]). The variation in the centroid is approximately proportional to the square root of intensity.<br />
<br />
As a result, the diffusion coefficient measured by taking intensity weighted centroids of a perfectly stationary particle will not be zero. The measured diffusion coefficient of a non stationary particle will be systematically increased.<br />
<br />
Here is the code that generated the simulated images:<br />
<pre><br />
figure<br />
<br />
maximumIntensity = [1e6 1e4 1e2 1e1 1e0];<br />
radius = 10;<br />
imageSize = [40 40];<br />
<br />
for ii = 1:length(maximumIntensity)<br />
subplot(1, length(maximumIntensity), ii)<br />
imshow(poissrnd(SimulateFluorescentParticleImage(imageSize ./ 2, maximumIntensity(ii), radius, imageSize)) ...<br />
./ maximumIntensity(ii));<br />
title(['SNR = ' num2str(sqrt(maximumIntensity(ii)))]);<br />
end<br />
<br />
<br />
function [ OutputImage ] = SimulateFluorescentParticleImage( CentroidList, Intensity, Raduis, ImageSize )<br />
<br />
numberOfParticles = size(CentroidList,1);<br />
<br />
OutputImage = zeros(ImageSize);<br />
<br />
if(length(Raduis) == 1)<br />
Radius = Raduis * ones(1, numberOfParticles);<br />
end<br />
<br />
if(length(Intensity) == 1)<br />
Intensity = Intensity * ones(1, numberOfParticles);<br />
end<br />
<br />
for ii=1:numberOfParticles<br />
OutputImage = OutputImage + DrawParticle(CentroidList(ii, 1), CentroidList(ii, 2), Radius(ii), Intensity(ii), ...<br />
ImageSize(1), ImageSize(2));<br />
end<br />
end<br />
<br />
<br />
</pre><br />
<br />
<References /><br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/Optical_Microscopy:_Part_4_Report_OutlineOptical Microscopy: Part 4 Report Outline2015-10-12T20:38:56Z<p>Steven Nagle: </p>
<hr />
<div><ol start="5"><br />
</li> <li>Viscosity <br />
<ol><br />
<li>Procedure<br />
<ul><li>Document the samples you prepared and used and how you captured images (camera settings including frame acquisition rate, number of frames, number of particles in the region of interest, choice of sample plane, etc)</li></ul><br />
</li><br />
<li>Data</li><br />
<ul><br />
<li>Include a snapshot of the 0.84 &mu;m fluorescent beads monitored.</li><br />
<li>Plot one or more example bead trajectories for each of the samples. Use of a similar scale for all plots would be informative to the reader.</li><br />
<li>Plot all of your MSD vs &tau; results; use log-log axes. Use the minimum number of axes that can convey your results clearly.</li><br />
</ul><br />
<li>Analysis and Results</li><br />
<ul><br />
<li>Include a table of the diffusion coefficient, viscosity and glycerin/water ratio for each of the samples (A, B, C, and D). All quantities must be reported with an associated uncertainty.</li><br />
<li>Plot the average MSD for all three PVP samples on a single set of log-log axes (0h, 1h, and 3h).</li><br />
<li>Provide a bullet point outline of all calculations and data processing steps.</li><br />
</ul><br />
<li>Discussion</li><br />
<ul><br />
<li>How do your viscosity calculations compare to your expectations? (This [https://dl.dropboxusercontent.com/u/12957607/Viscosity%20of%20Aqueous%20Glycerine%20Solutions.pdf chart] is a useful reference.)</li><br />
<li>What can you infer about the viscoelastic properties of the PVP gels from your MSD plots?</li><br />
<li>Include a thorough discussion of error sources and the approaches to minimize them.</li><br />
</ul><br />
</ol><br />
</li><br />
</li> <br />
</ol></div>Steven Naglehttp://measurebiology.org/wiki/Optical_Microscopy:_Part_4_Report_OutlineOptical Microscopy: Part 4 Report Outline2015-10-12T20:38:18Z<p>Steven Nagle: </p>
<hr />
<div><ol start="5"><br />
</li> <li>Viscosity <br />
<ol><br />
<li>Procedure<br />
<ul><li>Document the samples you prepared and used and how you captured images (camera settings including frame acquisition rate, number of frames, number of particles in the region of interest, choice of sample plane, etc)</li></ul><br />
</li><br />
<li>Data</li><br />
<ul><br />
<li>Include a snapshot of the 0.84 &mu;m fluorescent beads monitored.</li><br />
<li>Plot one or more example bead trajectories for each of the samples. Use of a similar scale for all plots would be informative to the reader.</li><br />
<li>Plot all of your MSD vs &tau results; use log-log axes. Use the minimum number of axes that can convey your results clearly.</li><br />
</ul><br />
<li>Analysis and Results</li><br />
<ul><br />
<li>Include a table of the diffusion coefficient, viscosity and glycerin/water ratio for each of the samples (A, B, C, and D). All quantities must be reported with an associated uncertainty.</li><br />
<li>Plot the average MSD for all three PVP samples on a single set of log-log axes (0h, 1h, and 3h).</li><br />
<li>Provide a bullet point outline of all calculations and data processing steps.</li><br />
</ul><br />
<li>Discussion</li><br />
<ul><br />
<li>How do your viscosity calculations compare to your expectations? (This [https://dl.dropboxusercontent.com/u/12957607/Viscosity%20of%20Aqueous%20Glycerine%20Solutions.pdf chart] is a useful reference.)</li><br />
<li>What can you infer about the viscoelastic properties of the PVP gels from your MSD plots?</li><br />
<li>Include a thorough discussion of error sources and the approaches to minimize them.</li><br />
</ul><br />
</ol><br />
</li><br />
</li> <br />
</ol></div>Steven Naglehttp://measurebiology.org/wiki/Optical_Microscopy:_Part_4_Report_OutlineOptical Microscopy: Part 4 Report Outline2015-10-12T20:35:03Z<p>Steven Nagle: </p>
<hr />
<div><ol start="5"><br />
</li> <li>Viscosity <br />
<ol><br />
<li>Procedure<br />
<ul><li>Document the samples you prepared and used and how you captured images (camera settings including frame acquisition rate, number of frames, number of particles in the region of interest, choice of sample plane, etc)</li></ul><br />
</li><br />
<li>Data</li><br />
<ul><br />
<li>Include a snapshot of the 0.84 &mu;m fluorescent beads monitored.</li><br />
<li>Plot one or more example bead trajectories for each of the samples. Use of a similar scale for all plots would be informative to the reader.</li><br />
<li>Plot all of your MSD vs &tau results; use log-log axes. Use the minimum number of axes that can convey your results clearly.</li><br />
</ul><br />
<li>Analysis and Results</li><br />
<ul><br />
<li>Include a table of the diffusion coefficient, viscosity and glycerin/water ratio for each of the samples (A, B, C, and D).</li><br />
<li>Plot the average MSD for all three PVP samples on a single set of log-log axes (0h, 1h, and 3h).</li><br />
<li>Provide a bullet point outline of all calculations and data processing steps.</li><br />
</ul><br />
<li>Discussion</li><br />
<ul><br />
<li>How do your viscosity calculations compare to your expectations? (This [https://dl.dropboxusercontent.com/u/12957607/Viscosity%20of%20Aqueous%20Glycerine%20Solutions.pdf chart] is a useful reference.)</li><br />
<li>What can you infer about the viscoelastic properties of the PVP gels from your MSD plots?</li><br />
<li>Include a thorough discussion of error sources and the approaches to minimize them.</li><br />
</ul><br />
</ol><br />
</li><br />
</li> <br />
</ol></div>Steven Naglehttp://measurebiology.org/wiki/Optical_Microscopy_Part_3:_Resolution_and_StabilityOptical Microscopy Part 3: Resolution and Stability2015-10-12T20:20:29Z<p>Steven Nagle: </p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:Optical Microscopy Lab]]<br />
{{Template:20.309}}<br />
<br />
==Overview==<br />
Congratulations on completing part 2. You made a functioning epifluorescence microscope. How cool is that?<br />
<br />
In this part of the lab, you will measure the resolution of your microscope using tiny, fluorescent microspheres as sources. Next, you will quantify the stability of your microscope by tracking immobile beads. In part 4 of this lab, you will characterize the diffusive motion of particles in different rheological environments by tracking fluorescent microspheres. Your microscope will act as a position detector. To prepare for these measurements, you will measure the position noise of your instrument to establish a baseline performance parameter.<br />
<br />
==Finding things==<br />
[[Image:Simulated Shot Noise.png|Simulated image of a fluorescent microshpere at various signal to noise ratios.]]<br />
Shot noise is a fluctuation that affects all light intensity measurements, including microscopic images recorded with a CCD camera. <ref>There are a few exotic methods, such as [http://www.rp-photonics.com/amplitude_squeezed_light.html amplitude-squeezed light] that reduce noise below the shot noise level.</ref> It is a consequence the discrete and the stochastic nature of photon emission. Poisson statistics provide an excellent model for shot noise. The standard deviation of a Poisson-distributed random variable is equal to the square root of its average value. Thus, the lowest possible signal to noise ratio of an intensity measurement is equal to the square root of the average number of photons (intensity). The simulated images above show the effect of shot noise on the image of a fluorescent microsphere with a radius of 10 pixels for several values of intensity. <br />
<br />
One method to estimate the position of a microsphere in an image is to compute an intensity-weighted centroid, also called the center of mass. Intensity-weighted centroids can provide locations accurate to a fraction of a pixel. Shot noise causes random variation in the pixel intensities, which perturbs the centroid. Repeated localizations of a stationary particle will exhibit random variation. Deriving an analytical expression for the variation is tedious. Happily, patient people with exceptional mathematical abilities have taken the time to do so. (See, for example, this reference: [http://www.opticsinfobase.org/josaa/abstract.cfm?uri=josaa-27-9-2038|Hui Jia, Jiankun Yang, and Xiujian Li. Minimum variance unbiased subpixel centroid estimation of point image limited by photon shot noise.]). The variation in the centroid is approximately proportional to the square root of intensity.<br />
<br />
As a result, a diffusion coefficient measured by repeatedly taking intensity weighted centroids of a perfectly stationary particle will not be zero. The measured diffusion coefficient of a non stationary particle will be systematically increased.<br />
<br />
Here is the code that generated the simulated images:<br />
<pre><br />
figure<br />
<br />
maximumIntensity = [1e6 1e4 1e2 1e1 1e0];<br />
radius = 10;<br />
imageSize = [40 40];<br />
<br />
for ii = 1:length(maximumIntensity)<br />
subplot(1, length(maximumIntensity), ii)<br />
imshow(poissrnd(SimulateFluorescentParticleImage(imageSize ./ 2, maximumIntensity(ii), ...<br />
radius, imageSize)) ./ maximumIntensity(ii));<br />
title(['SNR = ' num2str(sqrt(maximumIntensity(ii)))]);<br />
end<br />
<br />
<br />
function [ OutputImage ] = SimulateFluorescentParticleImage( CentroidList, Intensity, ...<br />
Raduis, ImageSize )<br />
<br />
numberOfParticles = size(CentroidList,1);<br />
<br />
OutputImage = zeros(ImageSize);<br />
<br />
if(length(Raduis) == 1)<br />
Radius = Raduis * ones(1, numberOfParticles);<br />
end<br />
<br />
if(length(Intensity) == 1)<br />
Intensity = Intensity * ones(1, numberOfParticles);<br />
end<br />
<br />
for ii=1:numberOfParticles<br />
OutputImage = OutputImage + DrawParticle(CentroidList(ii, 1), CentroidList(ii, 2), ...<br />
Radius(ii), Intensity(ii), ImageSize(1), ImageSize(2));<br />
end<br />
end<br />
<br />
<br />
</pre><br />
<br />
==Instructions==<br />
<br />
===Measuring resolution===<br />
<br />
One of the most commonly used definitions of resolution is the distance between two point sources in the sample plane such that the peak of one source’s image falls on the first minimum of the other source’s image. This suggests a procedure for measuring resolution: image a point source; measure the peak-to-trough distance; and divide by the magnification. In this part of the lab, you will use this method to estimate the resolution of your microscope.<br />
<br />
[[Image:20.309_140218_PSFbeads.png|frameless|thumb|Example image processing on PSF beads to determine microscope resolution.]]<br />
[[Image:20.309_140218_GaussianFit.png|frameless|thumb|Example Gaussian fit of a PSF bead fluorescence emission profile to estimate microscope resolution.]]<br />
<br />
A practical problem with this method is that true point sources are difficult to come by. If you are using a telescope, stars are readily available approximate point sources. In microscopy, people usually use tiny, fluorescent beads with diameters of 100-190 nm. These beads are small enough to be considered point sources. You will use nonlinear regression to estimate the resolution of your microscope from an image of the tiny beads. Unfortunately, beads small enough for this purpose are not very bright. Imaging them can be challenging. Your microscope must be aligned very well to get good results.<br />
<br />
You will use image processing functions to locate the beads in your image and fit a Gaussian function to them. Gaussians are more amenable to nonlinear regression than Bessel functions, and they are a very good approximation. It is straightforward to convert the Gaussian parameters to Rayleigh resolution. See [[Converting Gaussian fit to Rayleigh resolution]] for a discussion of the conversion.<br />
<br />
# Make an image of a sample of 170 nm fluorescent beads with the 40X objective. (Several dozens to hundreds of PSF spheres should be captured in your image.)<br />
#* Use 12-bit mode on the camera and make sure to save the image in a format that preserves all 12 bits.<br />
#* Use <code>imhist</code> to ensure that the image is exposed properly.<br />
#** Since there are a very small number of bright pixels, plot the histogram counts on a logarithmic scale.<br />
#* '''Include the image and the histogram in your lab report.'''<br />
# Use image processing functions to locate non-overlapping, single beads in the image.<br />
# Use nonlinear regression to fit a Gaussian to each bead image.<br />
# Convert the Gaussian parameters to resolution.<br />
# '''Report the results in your lab report.'''<br />
#* [[MATLAB: Estimating resolution from a PSF slide image|This page]] has example MATLAB code.<br />
#* '''Discuss how the measured resolution compares with the theoretical value.'''<br />
<br />
===Stability of microscope for particle tracking===<br />
<br />
The accuracy of optical particle tracking may be limited by mechanical and optical phenomena. Vibration and drift are a source of additive noise. Shot noise and CCD readout noise in the image of a particle bring about uncertainty in the estimate of its centroid. Excessive vibration can frequently be corrected by improving the mechanical support structure of the instrument. Most stages can be locked to reduce drift. Shot noise is fundamental; however, its relative contribution to the total signal can be minimized by ensuring that the optical system is functioning at peak efficiency.<br />
<br />
Before attempting to make measurements with particle tracking, it is essential to determine the performance characteristics of the instrument to be used. This can be accomplished by measuring a specimen with known characteristics. Perhaps the most foolproof choice is a sample with fixed particles. Any measured variation in the fixed sample is noise.<br />
<br />
* To verify that your system is sufficiently stable for accurate particle tracking, monitor a dry specimen containing 0.84&mu;m fluorescent beads. <br />
<br />
[[Image:Stability Plot.png|right|300px|thumb|Example stability plot from the demo microscope.]]<br />
<br />
# Bring a slide with fixed beads into focus. Choose a field of view in which you can see at least 3 beads with the 40× objective. Limit the field of view to only those beads by choosing a region of interest (ROI) in <tt>imaqtool</tt>.<br />
# Track the beads for 3 minutes in a Matlab video and save the centroids with a frame rate of your choice and make a note of it.<br />
# Use the Matlab function <code>track</code> (check out the Stellar Reading Materials sections for an example, and be sure to limit the algorithm to a small region of interest around the beads, otherwise Matlab will struggle!) to separate the centroids into individual trajectories, <math>\vec r_n(t)</math>, where <math>t = nT</math> and <math>T</math> is the inverse of the frame rate you set above.<br />
# Compute the difference of the trajectories for two particles, <math>\vec r_-(t) = {{\vec r_1(t) - \vec r_2(t)} \over \sqrt{2}}</math>. (Why is the square root of 2 necessary?)<br />
# Compute and plot the mean squared displacement (MSD) of <math>r_-</math> as a function of time interval and compare to the MSD of the actual particle tracks, <math>\left \langle {\left | \vec r(t+\tau)-\vec r(t) \right \vert}^2 \right \rangle</math> for intervals <math>\tau=nT</math> up to 180 s.<br />
<br />
* Why does the MSD of the particle trajectory increase while the difference trajectory stays about constant over the range of lag times &tau;? <br />
* Can you take advantage of this property to decrease the error in measurements of unknown samples?<br />
* Make any necessary adjustments to your microscope and repeat this particle-tracking procedure to attain sufficient stability:<br />
** The MSD from the difference trajectory should start out less than 100 nm<sup>2</sup> at t = 1 s and still be less than 1000 nm<sup>2</sup> for t = 180 s.<br />
<br />
<br />
<br />
==Report==<br />
<br />
Find and follow all guidelines on the [[Microscopy report outline]] wiki page. <br />
<br />
{{:Optical Microscopy: Part 3 Report Outline}} <br />
<br />
{{:Optical microscopy lab wiki pages}}<br />
<br />
==References==<br />
<br />
<References/><br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/MATLAB:_Estimating_resolution_from_a_PSF_slide_imageMATLAB: Estimating resolution from a PSF slide image2015-10-06T20:20:55Z<p>Steven Nagle: </p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:Matlab]]<br />
{{Template:20.309}}<br />
<br />
[[Image:Synthetic PSF Image.png|thumb|right|Synthetic PSF image generated with MATLAB.]]<br />
<br />
This page has example code for estimating resolution from an image of approximate point sources on a dark background, such as a star field or sub resolution fluorescent microspheres. <br />
<br />
==Measuring resolution==<br />
<tt>EstimateResolutionFromPsfImage</tt> takes a point-source image and estimates the resolution of an optical system. It uses the built-in MATLAB function <tt>[http://www.mathworks.com/help/images/ref/im2bw.html im2bw]</tt> to locate bright regions and <tt>[http://www.mathworks.com/help/images/ref/regionprops.html regionprops]</tt> to measure attributes of each connected region of bright pixels. After rejecting outliers, the function uses <tt>[http://www.mathworks.com/help/images/ref/nlinfit.html nlinfit]</tt> to estimate best fit Gaussian parameters for each bright spot. The optional second argument controls the rejection range for outliers.<br />
<br />
There are four subfunctions that should be included in the same m-file as EstimateResolutionFromPsfImage.<br />
<br />
<br />
<pre><br />
function [ Resolution, StandardError, BestFitData ] = EstimateResolutionFromPsfImage( ImageData, OutlierTolerancePercentage )<br />
<br />
if( nargin < 2 )<br />
OutlierTolerancePercentage = [ 0.7 1.3];<br />
end<br />
<br />
ImageData = im2double(ImageData);<br />
<br />
figure(1);<br />
imshow( ImageData );<br />
hold on;<br />
title( 'PSF Image' );<br />
<br />
% create bilevel image mapping bright regions<br />
thresholdLevel = 0.5 * ( max( ImageData(:) ) + median( ImageData(:) ) ); % this may not work in all cases<br />
binaryImage = im2bw( ImageData, thresholdLevel );<br />
% increase the size of each connected region using dilation<br />
dilatedImage = imdilate( binaryImage, strel( 'disk', 7, 0 ) ); <br />
% remove objects touching the edges<br />
dilatedImage = imclearborder( dilatedImage ); <br />
<br />
% assign a unique object number to each connected region of pixels<br />
labeledImage = bwlabel( dilatedImage ); <br />
<br />
% draw a red circle around labeled objects<br />
CircleObjectsInImage( labeledImage, [ 1 0 0 ] ); <br />
<br />
% compute the parameters of each object identified in the image<br />
objectProperties = regionprops( dilatedImage, ImageData, ...<br />
'Area', 'Centroid', 'PixelList', 'PixelValues', 'MaxIntensity' );<br />
<br />
% eliminate outliers -- PSF beads that are touching, aggregates, etc...<br />
% begin by eliminating objects whose areas are outliers, as occurs when<br />
% beads are too close<br />
medianArea = median( [ objectProperties.Area ] );<br />
outliers = [ objectProperties.Area ] > OutlierTolerancePercentage(2) * medianArea | [ objectProperties.Area ] < OutlierTolerancePercentage(1) * medianArea;<br />
<br />
if( sum( outliers ) > 0 )<br />
dilatedImage = RemoveObjectsFromImage( dilatedImage, objectProperties( outliers ) );<br />
objectProperties( outliers ) = []; % remove outliers<br />
end<br />
<br />
CircleObjectsInImage( dilatedImage, [ 1 1 0 ] );<br />
<br />
% next eliminate intensity outliers<br />
medianMaxIntensity = median( [ objectProperties.MaxIntensity ] );<br />
outliers = [ objectProperties.MaxIntensity ] > OutlierTolerancePercentage(2) * medianMaxIntensity | [ objectProperties.MaxIntensity ] < OutlierTolerancePercentage(1) * medianMaxIntensity;<br />
outliers = outliers | [ objectProperties.MaxIntensity ] > 0.995;<br />
<br />
if( sum( outliers ) > 0 )<br />
dilatedImage = RemoveObjectsFromImage( dilatedImage, objectProperties( outliers ) );<br />
objectProperties( outliers ) = [];<br />
end<br />
<br />
% circle all the remaining objects in green<br />
CircleObjectsInImage( dilatedImage, [ 0 1 0 ] );<br />
LabelObjectsInImage( objectProperties );<br />
hold off;<br />
<br />
BestFitData = cell(1, length(objectProperties));<br />
<br />
% use nlinfit to fit a Gaussian to each object<br />
for ii = 1:length(objectProperties)<br />
% initial guess for sigma based on area of bright spot<br />
maximumPixelValue = max( objectProperties(ii).PixelValues );<br />
darkPixelValue = median( objectProperties(ii).PixelValues );<br />
pixelCountAboveHalf = sum( objectProperties(ii).PixelValues > .5 * ( maximumPixelValue + darkPixelValue ) );<br />
sigmaInitialGuess = 0.8 * sqrt( pixelCountAboveHalf / 2 / pi / log(2) );<br />
<br />
initialGuesses = [ ...<br />
objectProperties(ii).Centroid(1), ... % yCenter<br />
objectProperties(ii).Centroid(2), ... % xCenter<br />
max(objectProperties(ii).PixelValues) - min(objectProperties(ii).PixelValues), ... % amplitude<br />
sigmaInitialGuess, ... % (objectProperties(ii).BoundingBox(3) - 6) / 4, ... % sigma<br />
min(objectProperties(ii).PixelValues) ];<br />
<br />
BestFitData{ii} = nlinfit( objectProperties(ii).PixelList, objectProperties(ii).PixelValues, @Gaussian2DFitFunction, initialGuesses );<br />
<br />
% plot data, initial guess, and fit for each peak<br />
figure(2)<br />
clf<br />
<br />
% generate a triangle mesh from the best fit solution found by <br />
% nlinfit and plot it<br />
gd = delaunay( objectProperties(ii).PixelList(:,1), ...<br />
objectProperties(ii).PixelList(:,2) );<br />
trimesh( gd, objectProperties(ii).PixelList(:,1), ...<br />
objectProperties(ii).PixelList(:,2), ...<br />
Gaussian2DFitFunction(BestFitData{ii}, ...<br />
objectProperties(ii).PixelList ) )<br />
hold on<br />
<br />
% plot initial guesses -- commented out to make plots less<br />
% cluttered. put this back in to debug initial guesses<br />
% plot3( objectProperties(ii).PixelList(:,1), ...<br />
% objectProperties(ii).PixelList(:,2), ...<br />
% Gaussian2DFitFunction(initialGuesses, ...<br />
% objectProperties(ii).PixelList ), 'rx' )<br />
<br />
% plot image data<br />
plot3( objectProperties(ii).PixelList(:,1), ...<br />
objectProperties(ii).PixelList(:,2), ...<br />
objectProperties(ii).PixelValues, 'gx', 'LineWidth', 3)<br />
title(['Image data vs. Best Fit for Object Number ' num2str(ii)]);<br />
end<br />
<br />
allPeakData = vertcat( BestFitData{:} );<br />
Resolution = mean( allPeakData(:,4) ) ./ 0.336;<br />
StandardError = std( allPeakData(:,4) ./ 0.336 ) ./ sqrt( length( BestFitData ) );<br />
end<br />
<br />
function out = Gaussian2DFitFunction( Parameters, Coordinates )<br />
yCenter = Parameters(1);<br />
xCenter = Parameters(2);<br />
amplitude = Parameters(3);<br />
sigma = Parameters(4);<br />
offset = Parameters(5);<br />
<br />
out = amplitude * ...<br />
exp( -(( Coordinates(:, 1) - yCenter ).^2 + ( Coordinates(:, 2) - xCenter ).^2 ) ...<br />
./ (2 * sigma .^ 2 )) + offset;<br />
<br />
end<br />
<br />
function CircleObjectsInImage( LabelImage, BorderColor )<br />
boundaries = bwboundaries( LabelImage ); <br />
numberOfBoundaries = size( boundaries );<br />
for k = 1 : numberOfBoundaries<br />
thisBoundary = boundaries{k};<br />
plot(thisBoundary(:,2), thisBoundary(:,1), 'Color', BorderColor, 'LineWidth', 2);<br />
end<br />
end<br />
<br />
function LabelObjectsInImage( ObjectProperties )<br />
labelShift = -9;<br />
fontSize = 10;<br />
<br />
for ii = 1:length(ObjectProperties)<br />
unweightedCentroid = ObjectProperties(ii).Centroid; % Get centroid.<br />
text(unweightedCentroid(1) + labelShift, unweightedCentroid(2), ...<br />
num2str(ii), 'FontSize', fontSize, 'HorizontalAlignment', ...<br />
'Right', 'Color', [0 1 0]);<br />
end<br />
end<br />
<br />
function OutputBinaryImage = RemoveObjectsFromImage( InputBinaryImage, ObjectProperties )<br />
OutputBinaryImage = InputBinaryImage;<br />
eliminatedPixels = vertcat( ObjectProperties.PixelList );<br />
allObjectIndexes = sub2ind( size( InputBinaryImage ), ...<br />
eliminatedPixels(:, 2), eliminatedPixels(:,1) );<br />
OutputBinaryImage( allObjectIndexes ) = 0;<br />
end<br />
<br />
% initial version 9/23/2013 by SCW<br />
</pre><br />
<br />
==Testing the code==<br />
It is an excellent idea to test functions before using them. One way to test <tt>EstimateResolutionFromPsfImage</tt> is to generate a synthetic point-source image of known resolution. <br />
<br />
===Generating a synthetic PSF image===<br />
<tt>SimulatePsfSlide</tt> repeatedly calls subfunction <tt>SimulateAiryDiskImage</tt> to generate a synthetic PSF slide image.<br />
<br />
<pre><br />
function SimulatedImage = SimulatePsfSlide( ...<br />
NumericalAperture, Magnification, Wavelength, ...<br />
Intensity, PixelSize, ImageSize, NumberOfBeads, ...<br />
ProbabilityOfAggregation, TechnicalNoise, ElectronsPerCount )<br />
<br />
resolution = 0.61 * Wavelength / NumericalAperture;<br />
airyDiskRadiusPixels = resolution * Magnification / PixelSize;<br />
<br />
SimulatedImage = zeros( ImageSize );<br />
<br />
for ii = 1:NumberOfBeads<br />
beadCenter = rand( 1, 2 ) .* ImageSize;<br />
intensity = ( 1 + 0.1 * randn() ) * Intensity * ( 1 + poissrnd(ProbabilityOfAggregation) );<br />
SimulatedImage = SimulatedImage + ...<br />
SimulateAiryDiskImage( beadCenter, airyDiskRadiusPixels, ImageSize, intensity );<br />
end<br />
<br />
SimulatedImage = SimulatedImage + TechnicalNoise .* randn( ImageSize );<br />
SimulatedImage = uint16( round( SimulatedImage ./ ElectronsPerCount ) );<br />
end<br />
<br />
function out = SimulateAiryDiskImage( AiryDiskCenter, AiryDiskRadius, ImageSize, Intensity )<br />
<br />
yAxis = (1:ImageSize(1)) - AiryDiskCenter(1);<br />
xAxis = (1:ImageSize(2)) - AiryDiskCenter(2);<br />
<br />
[ xCoordinate, yCoordinate ] = meshgrid( xAxis, yAxis );<br />
<br />
firstBesselZero = 3.8317;<br />
normalizedRadius = firstBesselZero .* sqrt( xCoordinate.^2 + yCoordinate.^2 ) ./ AiryDiskRadius;<br />
<br />
out = Intensity .* ( 2 * besselj(1, normalizedRadius ) ./ ( normalizedRadius ) ) .^ 2;<br />
<br />
% remove NaN from result if center is an integer<br />
if( all(AiryDiskCenter == fix( AiryDiskCenter )) )<br />
out( AiryDiskCenter(1), AiryDiskCenter(2) ) = Intensity;<br />
end<br />
end<br />
<br />
</pre><br />
<br />
===Testing the code===<br />
The script below generates synthetic images over a range of resolutions and compares them to the expected results.<br />
[[Image:Code Test Results.png|thumb|right|Code testing results.]]<br />
<br />
<pre><br />
<br />
naValues = 0.65:.025:1.25;<br />
figureHandle = figure();<br />
<br />
measuredResolution = zeros( 1, length(naValues) );<br />
theoreticalResolution = zeros( 1, length(naValues) );<br />
standardError = zeros( 1, length(naValues) );<br />
<br />
for ii=1:length(naValues)<br />
% generate synthetic PSF image<br />
NumericalAperture = naValues(ii); %0.5; %65;<br />
Magnification = 40;<br />
Wavelength = 590e-9; % m<br />
ProbabilityOfAggregation = 0; %0.2;<br />
PixelSize = 7.4e-6; % m<br />
ImageSize = [500 500]; % pixels x pixels<br />
NumberOfBeads = 100;<br />
TechnicalNoise = 0; % electrons per exposure<br />
ElectronsPerCount = 2; <br />
Intensity = 4095 * ElectronsPerCount * 0.8; % photons per exposure at peak<br />
<br />
simulatedPsfImage = SimulatePsfSlide( ...<br />
NumericalAperture, Magnification, Wavelength, ...<br />
Intensity, PixelSize, ImageSize, NumberOfBeads, ...<br />
ProbabilityOfAggregation, TechnicalNoise, ElectronsPerCount );<br />
<br />
% detect and simulate clipping due to overexposure<br />
if( max( simulatedPsfImage(:) ) > 4095 )<br />
disp('*** Overexposed Image ***');<br />
simulatedPsfImage( simulatedPsfImage > 4095 ) = 4095;<br />
end<br />
<br />
simulatedPsfImage = im2double( simulatedPsfImage * 16 ); <br />
<br />
theoreticalResolution(ii) = 0.61 * Wavelength / NumericalAperture;<br />
<br />
[ measuredResolution(ii), standardError(ii), bestFitData ] = ...<br />
EstimateResolutionFromPsfImage( simulatedPsfImage );<br />
<br />
measuredResolution(ii) = measuredResolution(ii) .* PixelSize;<br />
standardError(ii) = standardError(ii) .* PixelSize;<br />
<br />
figure( figureHandle );<br />
plot( theoreticalResolution(1:ii), measuredResolution(1:ii) );<br />
errorbar( theoreticalResolution(1:ii), measuredResolution(1:ii), standardError(1:ii) );<br />
title( 'Measured vs. Theoretical Resolution' );<br />
xlabel( 'Theoretical Resolutuion' );<br />
ylabel( 'Measured Resolution' );<br />
<br />
end<br />
<br />
% initial version 9/23/2013 by SCW<br />
</pre><br />
<br />
===Making the simulation more realistic &mdash; adding noise.===<br />
The simulation is unrealistic in several ways. Add models for noise, optical aberrations, nonuniform illumination, etc... to characterize the effect of nonidealities on measurement accuracy and uncertainty.<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/Optical_Microscopy:_Part_2_Report_OutlineOptical Microscopy: Part 2 Report Outline2015-09-30T18:29:21Z<p>Steven Nagle: </p>
<hr />
<div># Microscope documentation<br />
## Include an updated block diagram of your microscope.<br />
# Images<br />
## Include a figure with an images of the 3.26 &mu;m fluorescent microsphere samples, and the stained cell samples with and without Cyto-D. <br />
##* For each sample, create 1 figure with 5 panels.<br />
##* The panels of the figure should be: A) unprocessed image; B) reference image; C) dark image; D) flat-field corrected image; and E) histogram. <br />
##* In the caption, specify the exposure and gain settings. Each image should have a scale bar. State the dimension of the scale bar in the caption.<br />
##* For panel E, plot histograms of the unprocessed, dark, reference, and corrected image on the same set of axes. Plot <tt>log10( count )</tt> on the vertical axis and intensity on the horizontal axis. Use a line plot instead of a bar chart for the histogram.<br />
## Image profile<br />
##* For one reference, dark and cell image set, plot an intensity profile across the same diagonal. You may also use a bead image, along with it's unique reference and dark images. The intensity of your three images should be on the same scale, i.e., 0 to 65,535 or 0 to 1. Place all three profiles on a single set of axes for comparison. (Use the <tt>improfile</tt> command in MATLAB.)<br />
# Discussion<br />
## How did your beam expander design affect your images?<br />
## What differences did you observe between the cells with and without CytoD?</div>Steven Naglehttp://measurebiology.org/wiki/Optical_microscopy_lab_wiki_pagesOptical microscopy lab wiki pages2015-09-24T16:23:46Z<p>Steven Nagle: /* Background reading */</p>
<hr />
<div>==Optical microscopy lab==<br />
===Lab manuals===<br />
* [[Optics Bootcamp]]<br />
* [[Lab Manual: Optical Microscopy]]<br />
* [[Optical Microscopy Part 1: Brightfield Microscopy]]<br />
* [[Optical Microscopy Part 2: Fluorescence Microscopy]]<br />
* [[Optical Microscopy Part 3: Resolution and Stability]]<br />
* [[Optical Microscopy Part 4: Particle Tracking]]<br />
* [[Microscopy report outline|Optical Microscopy Report Outline]]<br />
<br />
===Code examples and simulations===<br />
* [[Converting Gaussian fit to Rayleigh resolution]]<br />
* [[MATLAB: Estimating resolution from a PSF slide image]]<br />
* [[Matlab: Scalebars]]<br />
<br />
===Background reading===<br />
* [[Geometrical optics and ray tracing]]<br />
* [[Physical optics and resolution]]<br />
* [[Optical aberrations]]<br />
* [[Aperture and field stops]]<br />
* [[Optical detectors, noise, and the limit of detection]]<br />
* [[Manta G032 camera measurements]]</div>Steven Naglehttp://measurebiology.org/wiki/Manta_G032_camera_measurementsManta G032 camera measurements2015-09-23T00:13:08Z<p>Steven Nagle: /* Calculations */</p>
<hr />
<div>{{Template:20.309}}<br />
<br />
==Overview==<br />
This page contains data from the demo I did in lecture on 9/22/2015 of the Manta. The point of the demo was to measure the gain <math>g</math>, dark current <math>i_d</math>, and read noise <math>N_r</math> of the Manta G032 cameras we use in the microscopy lab. Note that I showed a linear plot in lecture and the plot below is log-log.<br />
<br />
* Gain relates the binary value reported by the camera to the number of electrons collected in a pixel: <math>P_{x,y}=g N_{x,y}</math>, where <math>P_{x,y}</math> is the value reported by the camera at pixel location <math>x</math>, <math>y</math>, and <math>N_{x,y}</math> is the number of electrons detected.<br />
* Dark current is the average number of dark electrons that are collected in units of electrons per second.<br />
* Read noise is a roughly Gaussian distributed random variable that lumps together noise sources that arise when counting electrons.<br />
<br />
==Measurement procedure==<br />
[[File:Manta Noise Measurement.png|right|thumb|400 px]]<br />
* Direct a light source at the camera to produce a range of intensities on the surface of the detector.<br />
* Record a 100 frame movie of the light source at 20 FPS with an exposure of 150 &mu;s.<br />
* Turn off the light source and record a 100 frame dark movie with identical exposure settings.<br />
* Compute the dark image by averaging all frames of the dark movie.<br />
* Subtract the dark image from each frame of the light movie.<br />
* Compute the variance of each pixel (noise squared) and plot versus the average value (signal).<br />
<br />
==Calculations==<br />
The value of a particular pixel over a certain time interval, <math>P_{x,y}[t]</math>, is equal to the sum of the number of photoelectrons plus the number of dark electrons plus the number of electrons gained or lost due to read noise. (The square brackets indicate that <math>P_{x,y}</math> is evaluated at discrete time points.) Mathematically:<br />
<br />
:<math>P_{x,y}[t]=g \left(I_{x,y}[t]+R_{x,y}[t]+D_{x,y}(t)) \right)</math>,<br />
where<br />
* <math>I_{x,y}[t]</math> is the number of photoelectrons generated during interval <math>t</math>,<br />
* <math>R_{x,y}[t]</math> is the read noise during time interval <math>t</math>,<br />
* and <math>D_{x,y}[t]</math> is the number of dark current electrons generated during time interval <math>t</math>.<br />
<br />
The next step is to write an expression for the mean value of each pixel. Means of terms in a sum add, so <math>\langle (P_{x,y}) \rangle</math> can be found by summing the means of the three individual terms. The mean value of read noise is zero, and the mean value of dark current is <math>i_d \delta t</math>, which gives:<br />
<br />
:<math>\langle P_{x,y}\rangle = g\left(\langle I_{x,y}\rangle + i_d \delta t \right)</math><br />
<br />
We subtracted the average dark frame to remove <math>i_d \delta t</math>, so the value plotted on the horizontal axis is just <math>g\left(\langle I_{x,y}\rangle \right)</math>. Cool.<br />
<br />
Now we need an expression for the noise. Variances of a sum of terms also add, so <math>\operatorname{Var}(P_{x,y})</math> can be found by summing the variances of the three individual terms. The photoelectron count, <math>I_{x,y}</math>, is Poisson distributed, so its variance is equal to its mean: <math>\operatorname{Var}(I_{x,y})=\langle I_{x,y} \rangle</math>. The second term has a constant variance that is a property of the camera, the read noise <math>N_r</math>. The third term is also Poisson distributed, with an average value of <math>i_d \delta t</math>, where <math>\delta t</math> is the exposure time. This gives:<br />
<br />
:<math>\text{Var}\left(P_{x,y}\right)=g\left(\langle I_{x,y}\rangle+N_r^2 + i_d \delta t \right)</math><br />
<br />
The x-intercept is equal to read noise plus dark noise times <math>g</math>. The slope of the line is equal to <math>g</math>.<br />
<br />
==Results==<br />
The slope of the line = 0.2987, meaning that the camera produces approximately 1 count per 3.3 electrons.<br />
<br />
The intercept is around 15 counts, meaning that dark plus read noise is about 15 counts, or about 50 electrons for this exposure.<br />
<br />
I took another dataset at a different exposure time that will allow us to figure out the relative contributions of dark and read noise. I will post soon.<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/Manta_G032_camera_measurementsManta G032 camera measurements2015-09-23T00:07:08Z<p>Steven Nagle: /* Calculations */</p>
<hr />
<div>{{Template:20.309}}<br />
<br />
==Overview==<br />
This page contains data from the demo I did in lecture on 9/22/2015 of the Manta. The point of the demo was to measure the gain <math>g</math>, dark current <math>i_d</math>, and read noise <math>N_r</math> of the Manta G032 cameras we use in the microscopy lab. Note that I showed a linear plot in lecture and the plot below is log-log.<br />
<br />
* Gain relates the binary value reported by the camera to the number of electrons collected in a pixel: <math>P_{x,y}=g N_{x,y}</math>, where <math>P_{x,y}</math> is the value reported by the camera at pixel location <math>x</math>, <math>y</math>, and <math>N_{x,y}</math> is the number of electrons detected.<br />
* Dark current is the average number of dark electrons that are collected in units of electrons per second.<br />
* Read noise is a roughly Gaussian distributed random variable that lumps together noise sources that arise when counting electrons.<br />
<br />
==Measurement procedure==<br />
[[File:Manta Noise Measurement.png|right|thumb|400 px]]<br />
* Direct a light source at the camera to produce a range of intensities on the surface of the detector.<br />
* Record a 100 frame movie of the light source at 20 FPS with an exposure of 150 &mu;s.<br />
* Turn off the light source and record a 100 frame dark movie with identical exposure settings.<br />
* Compute the dark image by averaging all frames of the dark movie.<br />
* Subtract the dark image from each frame of the light movie.<br />
* Compute the variance of each pixel (noise squared) and plot versus the average value (signal).<br />
<br />
==Calculations==<br />
The value of a particular pixel over a certain time interval, <math>P_{x,y}[t]</math>, is equal to the sum of the number of photoelectrons plus the number of dark electrons plus the number of electrons gained or lost due to read noise. (The square brackets indicate that <math>P_{x,y}</math> is evaluated at discrete time points.) Mathematically:<br />
<br />
:<math>P_{x,y}[t]=g \left(I_{x,y}[t]+R_{x,y}[t]+D_{x,y}(t)) \right)</math>,<br />
where<br />
* <math>I_{x,y}[t]</math> is the number of photoelectrons generated during interval <math>t</math>,<br />
* <math>R_{x,y}[t]</math> is the read noise during time interval <math>t</math>,<br />
* and <math>D_{x,y}[t]</math> is the number of dark current electrons generated during time interval <math>t</math>.<br />
<br />
The next step is to write an expression for the mean value of each pixel. Means of terms in a sum add, so <math>\langle (P_{x,y}) \rangle</math> can be found by summing the means of the three individual terms. The mean value of read noise is zero, and the mean value of dark current is <math>i_d \delta t</math>, which gives:<br />
<br />
:<math>\langle P_{x,y}\rangle = g\left(\langle I_{x,y}\rangle + i_d \delta t \right)</math><br />
<br />
We subtracted the average dark frame to remove <math>i_d \delta t</math>, so the value plotted on the horizontal axis is just <math>g\left(\langle I_{x,y}\rangle \right)</math>. Cool.<br />
<br />
Now we need an expression for the noise. Variances of a sum of terms also add, so <math>\operatorname{Var}(P_{x,y})</math> can be found by summing the variances of the three individual terms. The photoelectron count, <math>I_{x,y}</math>, is Poisson distributed, so its variance is equal to its mean: <math>\operatorname{Var}(I_{x,y})=\langle I_{x,y} \rangle</math>. The second term has a constant variance that is a property of the camera, the read noise <math>N_r</math>. The third term is also Poisson distributed, with an average value of <math>I_d \delta t</math>, where <math>\delta t</math> is the exposure time. This gives:<br />
<br />
:<math>\text{Var}\left(P_{x,y}\right)=g\left(\langle I_{x,y}\rangle+N_r^2 + i_d \delta t \right)</math><br />
<br />
The x-intercept is equal to read noise plus dark noise times <math>g</math>. The slope of the line is equal to <math>g</math>.<br />
<br />
==Results==<br />
The slope of the line = 0.2987, meaning that the camera produces approximately 1 count per 3.3 electrons.<br />
<br />
The intercept is around 15 counts, meaning that dark plus read noise is about 15 counts, or about 50 electrons for this exposure.<br />
<br />
I took another dataset at a different exposure time that will allow us to figure out the relative contributions of dark and read noise. I will post soon.<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/Manta_G032_camera_measurementsManta G032 camera measurements2015-09-23T00:03:24Z<p>Steven Nagle: /* Measurement procedure */</p>
<hr />
<div>{{Template:20.309}}<br />
<br />
==Overview==<br />
This page contains data from the demo I did in lecture on 9/22/2015 of the Manta. The point of the demo was to measure the gain <math>g</math>, dark current <math>i_d</math>, and read noise <math>N_r</math> of the Manta G032 cameras we use in the microscopy lab. Note that I showed a linear plot in lecture and the plot below is log-log.<br />
<br />
* Gain relates the binary value reported by the camera to the number of electrons collected in a pixel: <math>P_{x,y}=g N_{x,y}</math>, where <math>P_{x,y}</math> is the value reported by the camera at pixel location <math>x</math>, <math>y</math>, and <math>N_{x,y}</math> is the number of electrons detected.<br />
* Dark current is the average number of dark electrons that are collected in units of electrons per second.<br />
* Read noise is a roughly Gaussian distributed random variable that lumps together noise sources that arise when counting electrons.<br />
<br />
==Measurement procedure==<br />
[[File:Manta Noise Measurement.png|right|thumb|400 px]]<br />
* Direct a light source at the camera to produce a range of intensities on the surface of the detector.<br />
* Record a 100 frame movie of the light source at 20 FPS with an exposure of 150 &mu;s.<br />
* Turn off the light source and record a 100 frame dark movie with identical exposure settings.<br />
* Compute the dark image by averaging all frames of the dark movie.<br />
* Subtract the dark image from each frame of the light movie.<br />
* Compute the variance of each pixel (noise squared) and plot versus the average value (signal).<br />
<br />
==Calculations==<br />
The value of a particular pixel over a certain time interval, <math>P_{x,y}[t]</math>, is equal to the sum of the number of photoelectrons plus the number of dark electrons plus the number of electrons gained or lost due to read noise. (The square brackets indicate that <math>P_{x,y}</math> is evaluated at discrete time points.) Mathematically:<br />
<br />
:<math>P_{x,y}[t]=g \left(I_{x,y}[t]+R_{x,y}[t]+D_{x,y}(t)) \right)</math>,<br />
where<br />
* <math>I_{x,y}[t]</math> is the number of photoelectrons generated during interval <math>t</math>,<br />
* <math>R_{x,y}[t]</math> is the read noise during time interval <math>t</math>,<br />
* and <math>D_{x,y}[t]</math> is the number of dark current electrons generated during time interval <math>t</math><br />
<br />
The next step is to write an expression for the mean value of each pixel. Means of terms in a sum add, so <math>\langle (P_{x,y}) \rangle</math> can be found by summing the means of the three individual terms. The mean value of read noise is zero, and the mean value of dark current is <math>i_d \delta t</math>, which gives:<br />
<br />
:<math>\langle P_{x,y}\rangle = g\left(\langle I_{x,y}\rangle + i_d \delta t \right)</math><br />
<br />
We subtracted the average dark frame to remove <math>i_d \delta t</math>, so the value plotted on the horizontal axis is just <math>g\left(\langle I_{x,y}\rangle \right)</math>. Cool.<br />
<br />
Now we need an expression for the noise. Variances of a sum of terms also add, so <math>\operatorname{Var}(P_{x,y})</math> can be found by summing the variances of the three individual terms. The photoelectron count, <math>I_{x,y}</math>, is Poisson distributed, so its variance is equal to its mean: <math>\operatorname{Var}(I_{x,y})=\langle I_{x,y} \rangle</math>. The second term has a constant variance that is a property of the camera, the read noise <math>N_r</math>. The third term is also Poisson distributed, with an average value of <math>I_d \delta t</math>, where <math>\delta t</math> is the exposure time. This gives:<br />
<br />
:<math>\text{Var}\left(P_{x,y}\right)=g\left(\langle I_{x,y}\rangle+N_r^2 + i_d \delta t \right)</math><br />
<br />
The x-intercept is equal to read noise plus dark noise. The slope of the line is equal to <math>g</math>.<br />
<br />
==Results==<br />
The slope of the line = 0.2987, meaning that the camera produces approximately 1 count per 3.3 electrons.<br />
<br />
The intercept is around 15 counts, meaning that dark plus read noise is about 15 counts, or about 50 electrons for this exposure.<br />
<br />
I took another dataset at a different exposure time that will allow us to figure out the relative contributions of dark and read noise. I will post soon.<br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/DNA_melting_lab_wiki_pagesDNA melting lab wiki pages2015-09-22T19:33:29Z<p>Steven Nagle: /* Lab manuals */</p>
<hr />
<div>==DNA melting lab==<br />
===Lab manuals===<br />
* [[Lab Manual:Measuring DNA Melting Curves]]<br />
* [[DNA Melting Part 1: Measuring Temperature and Fluorescence]]<br />
* [[DNA Melting Report Requirements for Part 1]]<br />
* [[DNA Melting: Model function and parameter estimation by nonlinear regression]]<br />
* [[DNA Melting: Simulating DNA Melting - Intermediate Topics]]<br />
* [[DNA Melting Part 2: Lock-in Amplifier and Temperature Control]]<br />
* [[DNA Melting Report Requirements for Part 2]]<br />
<br />
===Code examples and simulations===<br />
* [[DNA Melting: Simulating DNA Melting - Basics]]<br />
* [[DNA Melting: Simulating DNA Melting - Intermediate Topics]]<br />
* [[DNA Melting: Model function and parameter estimation by nonlinear regression]]<br />
<br />
===Reference materials===<br />
* [[DNA Melting Thermodynamics]]<br />
* [[DNA Melting: DNA Sequences]]<br />
* [http://mfold.rna.albany.edu/?q=DINAMelt/Hybrid2 DINAMelt Web Server]<br />
<br />
===Background reading===<br />
* [[Electronics Primer]]<br />
<!-- * [[Real electronics]] --></div>Steven Naglehttp://measurebiology.org/wiki/Optical_Microscopy_Part_1:_Brightfield_MicroscopyOptical Microscopy Part 1: Brightfield Microscopy2015-09-22T19:32:38Z<p>Steven Nagle: /* Practice */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:Optical Microscopy Lab]]<br />
{{Template:20.309}}<br />
<br />
<blockquote><br />
<div><br />
''These I mention, that I may excite the World to enquire a little farther into the improvement of Sciences, and not think that either they or their predecessors have attained the utmost perfections of any one part of knowledge, and to throw off that lazy and pernicious principle, of being contented to know as much as their Fathers, Grandfathers, or great Grandfathers ever did, and to think they know enough, because they know somewhat more than the generality of the World besides:…Let us see what the improvement of Instruments can produce.''<br />
<blockquote><br />
''&mdash;Animadversions on the Machina Coelestis of Johannes Hevelius, 1674''<br />
</blockquote><br />
</div><br />
</blockquote><br />
<br />
<blockquote><br />
<div><br />
''Don't you just '''buy''' a [http://www.historycommons.org/context.jsp?item=a043074editedtranscripts [expletive deleted]] microscope?''<br />
<br />
<blockquote><br />
''&mdash;[http://web.mit.edu/fll/www/events/AforE/images/AEWinner1.jpg Anonymous 20.309 student], Fall 2007''<br />
</blockquote><br />
</div><br />
</blockquote><br />
<br />
==Overview==<br />
In the first week of the microscopy lab, you will construct a brightfield microscope.<br />
<br />
====Background materials and references==== <br />
<br />
The following online materials provide useful background for this part of the microscopy lab.<br />
<br />
* [[Geometrical optics and ray tracing]]<br />
* [[Physical optics and resolution]]<br />
* [https://stellar.mit.edu/S/course/20/fa13/20.309/materials.html Lectures 1 through 9 of the 20.309 class]<br />
* From [http://www.microscopyu.com Nikon MicroscopyU]<br />
** [http://www.microscopyu.com/articles/formulas/formulasconjugate.html Conjugate planes in optical microscopy] (includes transmitted and reflected (epi) illumination)<br />
** [http://www.microscopyu.com/articles/formulas/formulasri.html Snell's law]<br />
** [http://www.microscopyu.com/articles/formulas/formulasresolution.html Resolution]<br />
<br />
===Microscope block diagram===<br />
<br />
[[Image:20.309 130911 YourMicroscope.png|center|thumb|400px|20.309 microscope block diagram]]<br />
<br />
==Optical construction==<br />
Once you have settled on an arrangement of lenses and filters, the next challenge is to construct a system that will hold all the optics in their proper places and allow you to precisely locate a sample in front of the objective. Some of the optics must be very placed very accurately, while others don't matter so much. The position and angle of some components must be adjustable. The structure should be very rigid so that vibration does not degrade your images. <br />
<br />
There are several systems for optical construction available based on rails, posts, cages, tubes, and all manner of little, metallic bits. The 20.309 microscope is constructed chiefly from cage and lens tube components made by a company called [http://www.thorlabs.com/ ThorLabs]. Understanding how all of the components in the catalog work together is daunting. Ask about any components that perplex you.<br />
<br />
====Lenses====<br />
<br />
Plano-convex spherical lenses are available with focal lengths of 25, 50, 75, 100, 125, 150, 175, and 200 mm. Plano-concave lenses with focal lengths of -30 and -50 are also available. It is best to mount most optics in short (''e.g.'' 0.5") lens tubes. It is acceptable to mount a lens between the end of a tube and a tube ring or between two tube rings. In most cases, the convex side of the lens faces toward the collimated beam; the planar side goes toward the convergent rays. <br />
<br />
* ''Tip:'' Verify all optics before you use them by determining the focal length with a ruler. Use the ceiling fluorescent lamps as a light source and measuring the exact distance between the lens(es) assessed and the lamp's image. Can you imagine a simple rig to evaluate negative focal lengths (of plano-concave lenses for instance)?<br />
* ''Tip:'' As you install lenses into your microscope, put a piece of tape on the lens tube showing focal length and orientation. This will help you both during construction and put-away. Save the lens storage boxes and return components to the correct boxes when you are done.<br />
* Handle lenses only by the edges. If a lens is dirty, first remove grit with a blast of clean air or CO<sub>2</sub>. Clean the lens by wiping with a folded piece of lens paper wetted with a drop of methanol. (Do not touch the part of the tissue you use for cleaning with your fingers.) In some cases, it may be helpful to hold the folded lens tissue in a hemostat. Ask an instructor if you need help.<br />
<br />
====Objective lenses====<br />
<br />
Please see the Nikon [http://www.microscopyu.com/articles/optics/objectiveintro.html Introduction to Microscope Objectives] at their excellent [http://www.microscopyu.com/index.html MicroscopyU] website.<br />
<br />
There are three objective lenses available in the lab: a 10×, a 40×, and a 100×. All of these are designed for a 200 mm tube lens. An adapter ring converts the objective mounting threads to the SM1 threads used by the lens tube system.<br />
<br />
[[Image:20.309_130813_SimpleMicroscopeDiagram.png|center|thumb|400px|The reference tube length for the Nikon objectives we will use is 200 mm. A 200 mm lens, placed 200 mm from the back ring of the objective, will produce the rated magnification M.]]<br />
* The back focal plane (BFP) of the objective coincides with the rear of the objective housing. This is equivalent to the focal plane of a simple lens.<br />
* ''Working distance'' (WD) is the distance between the front end of the objective and the sample plane (when the sample is in focus). Generally, the higher the magnification, the lower the working distance.<br />
* The 100× objective is designed to be used with immersion oil, which provides an optical medium of pre-determined refractive index (''n'' = 1.5). When using the 100× objective, place a drop of oil on it. Bring the drop in contact with the slide cover glass. After use, clean off excess oil by wicking it away with lens paper. Do not put samples away dirty. It is not necessary to use immersion oil for thin samples such as the Air Force Target or Ronchi Ruling.<br />
<br />
====Sample stage====<br />
<br />
A precision Newport X/Y/Z stage<ref>[http://www.newport.com/562-Series-ULTRAlign-Precision-Multi-Axis-Positio/140089/1033/catalog.aspx Precision Newport X/Y/Z stages]</ref> with a sample holder mounted on a post, or a Thorlabs Max312D stage, also with a sample holder, is available at each lab station. The Newport stage setup is top-heavy. Avoid accidents by ensuring that the post base is always attached to an optical breadboard or table. Leave the stage at the lab station when you are done with it. For the Thorlabs stages, it is still a good idea to bolt them down so that your area of interest (AOI) stays in your microscope field of view (FOV).<br />
<br />
All stage axes have limited adjustment range, especially the Thorlabs stages. To deal with this, it is best to leave the stage base bolts and sample holder bolts loose and move the sample holder in x, y and z to roughly find your AOI. Once you are on or near your AOI, tighten the bolts and use the micrometers to center your image. One trick here is to get the z clamped first, then deal with x and y.<br />
<br />
====CCD camera====<br />
<br />
The microscope you will build does not have an eyepiece for direct visual observation. Instead, images will be captured with a CCD camera<ref>[http://www.alliedvisiontec.com/emea/products/cameras/gigabit-ethernet/manta/g-032bc.html Allied Manta G032B]</ref>. Its monochrome (black and white) sensor contains a grid of 656×492 square pixels that measure 7.4 μm on a side. An adapter ring converts the C-mount thread on the camera to SM1.<br />
<br />
==Microscope construction==<br />
<br />
===Design===<br />
<br />
Sketch out a rough design for your microscope on paper. Begin with the bright field illumination path.<br />
* Some elements must be positioned precise distances apart; other distances are not critical. Use ray-tracing to determine when this is the case. Which distances in your bright-field microscope will be critical? Which will be forgiving or unessential? Which will change with each objective lens (10×, 40× and 100×)?<br />
* Which sections of the light path can be open (strut-based structure)? Which would better enclosed (Thorlabs tubes)?<br />
* In what way will the illumination LED color affect your design? your results?<br />
* Which lens will you use between the LED and the sample for bright-field transmitted light imaging?<br />
<br />
===Practice===<br />
<br />
* Please do not remove parts from the example microscope.<br />
* On a 1' x 2' x <sup>1</sup>/<sub>2</sub>" optical breadboard, build a bright-field imaging microscope, using the provided LED as a light source, CCD camera as a light detector, and the rigid mounting components and lenses at your disposal.<br />
* Even though you're first focusing on the bright-field imaging leg of your microscope, take into consideration some requirements pertinent to the fluorescence imaging elements you'll add to your system next week:<br />
** Reproduce the general layout of the example microscope: it grants compactness and allows your device to be a stand-alone breadboard-transportable microscope. [[Image:130814_Microscope_All.jpg|center|thumb|400px|The general layout of the 20.309 microscope is compact and stand-alone; it fits and can be transported onto a breadboard]]<br />
** Do insert the C6W cage cube that will later hold the dichroic mirror on while fluorescence imaging will rely. Be sure to keep the mounting struts fully recessed in the cube walls; their ends should not stick out, they would otherwise hinder maneuvers with dichroic-holding kinematic plate!<br />
[[Image:130816_CageCube.png|center|thumb|400px|The mounting struts should remain recessed within the cage cube walls.]]<br />
* Set the distance between the top of the breadboard and the top of the upper LCP01 to '''13.5 cm'''. It is important to ensure your construction is compatible with either of the two distinct stage mounting platforms available in the 20.309 lab (either Newport or Thorlabs model). If you find it inconvenient to measure this, there is a Handy Scope Height Thingama-jig floating around the lab. Ask your instructor(s). Also, note that the stages are very expensive; always lift them from the bottom.<br />
* Verify the focal length of the lenses you selected. If you find an optic in the wrong box: identify the optic and replace it in the correct box or label the box correctly. (Ask an instructor if you can't find the right box. There are many boxes near the wire spools behind you as you stand at the wet bench.)<br />
* Check all your lenses for cleanliness before you use them. You'll save yourself some troubleshooting time and effort down the road!<br />
* Make sure all your components are "leveled" (horizontal, not slanted).<br />
* Use tube rings (and never an SM1T2, SM1V01, or SM1V05) to mount optics in lens tubes.<br />
* Use adjustable mounting components in front of the CCD camera so you can optimize and fine-tune the camera positing with respect to the imaging lens ''L2''. Beware: never use an SM1T2 coupler without a locking ring &mdash; they are very difficult to remove if they are tightened against a lens tube or tube ring. Also put a quick-connect in your design such that the camera CCD will end up 200 mm from the back focal plane of the objective. Remember that the CCD is recessed inside the opening of the camera.<br />
[[Image:20.309 130816 CCD QuickConnect.png|center|thumb|400px|Adjustable Thorlabs SM1V05 and SM1T2 connectors precede the quick-connect union to the CCD camera.]]<br />
* Restrict to 3 struts only the connection between the cage cube and the last silver mirror before the CCD camera, so you can easily take in and out the barrier filter ''BF'' that will later aid fluorescence-mode microscopy.<br />
[[Image:20.309 130816 BarrierFilterSpace.png|center|thumb|250px| Insertion and removal of optical components is facilitated by a three-strut-only link. ]]<br />
<br />
* The Nikon objective lenses are designed to be paired with a 200 mm tube lens.<br />
* Assume that the objectives behave as ideal plano-convex lenses.<br />
* Fine focusing will be achieved by adjusting the height of the sample stage.<br />
* ''Tip:'' Throughout the optical microscopy lab, start the alignment with a 10× objective but progress to 40× and 100×.<br />
* You can use either a red or a blue LED illuminator for bright-field transmitted light imaging. <br />
** Each group will receive their own LED. Please ask an instructor if you cannot find one.<br />
{{Template:Safety Warning|message=Double check your wiring before powering the LED. The LED can be damaged by excessive current. Limit the driving current to 0.5 A to protect the LED.}}<br />
<br />
==Magnification measurement==<br />
[[Image:20.309_130813_BrightFieldExampleImages.png|right|thumb|Example images included by past students in their Week 1 report: (top) Air Force target, (center) Silica spheres and dust, (bottom) Ronchi Ruling]]<br />
Measuring the magnification of your microscope is a good way to verify that your instrument is functioning well. You should measure the magnification of any microscope you plan to use for making quantitative measurements of size. Use the measured value in your calculations, not the number printed on the objective. Consider the uncertainty in your measurement.<br />
<br />
# Use MATLAB's Image Acquisition Tool to view a live display and record images<br />
#* Launch Matlab and type <tt>imaqtool</tt>. An Image Acquisition Tool window should fill the screen.<br />
#* Select the "Mono12" mode of the "Manta_G-032B (gentl-1)" camera in the Hardware Browser pane.<br />
#* Once it behaves, this setting will configure the camera to produce 12-bit, monochrome images. In this mode, the intensity of each pixel in the image will be represented by 12 binary digits, allowing a range of values from 0-4095.<br />
#* In the Acquisition Parameters pane, select the "Device Properties" tab and set "Acquisition Frame Rate Abs" to 20. This will cause the camera to take 20 complete images per second.<br />
#* Click the "Start Preview" button. The live image from the camera should appear in the Preview pane.<br />
#* If this does not produce a live image, close the window, issue the command imaqreset in the Matlab workspace. Then issue the command imaqtool again, choose the "gige-1" driver and start it as above. Regardless of whether it works, now close the window, issue the imaqreset command again, then the imaqtool command, and continue by choosing the "gentl-1" option. The drivers are, it goes without saying, a little flaky. It is best to avoid the "gige-1" option for long-running because it is more prone to hanging up than the "gentl-1" option.<br />
#* Use the "Exposure Auto" and "Exposure Time Abs" settings in the "Device Properties" tab to produce a good image. Setting "Exposure Auto" to "Once" will cause the camera to run its automatic exposure algorithm one time. This usually results in an exposure that's in the ballpark. But the automatic exposure usually does a poor job on microscopic images. Make the exposure better by changing the value in "Exposure Time Abs". The value sets the exposure time for each frame in microseconds.<br />
# Ensure that the camera's field of view is approximately centered in the objective's field of view. <br />
#* The objective has a larger FOV than the camera. Use the adjustment knobs on mirror M1 to traverse the objective's FOV horizontally and vertically. The FOV is approximately circular. Find a spot near the middle.<br />
# Measure the magnification of your microscope using the 10x, 40x, and 100x objectives.<br />
#* Start with the 10x objective and an Air Force imaging target.<br />
#** There are two styles of Air Force imaging target available in the lab. Both have sets of precisely spaced vertical and horizontal, high-contrast black/white line pairs. One version of the target indicates the size of the line pairs with a number conveniently printed near the set of lines. The number indicates how many black/white line pairs per millimeter. The other style of Air Force imaging target uses an annoying group/element designation that is explained on [[http://en.wikipedia.org/wiki/1951_USAF_resolution_test_chart this Wikipedia page]]. <br />
#** Make sure that the side of the target with the pattern on it faces the objective. Imaging through the thick glass causes distortion and many other troubles.<br />
#* Record an image of appropriately sized lines on the Air Force imaging target (with 10x and 40x objectives) and the Ronchi ruling (with the 100x objective).<br />
#** Why is it not judicious to image the Ronchi ruling with the 10x objective?<br />
#* Set "Frames per trigger" setting in the "General" tab of the Acquisition Parameters pane to 1. This setting controls how many images MATLAB will record each time you press the "Start Acquisition" button. <br />
#* Press the "Start Acquisition" in the Preview Pane. (The live preview will stop.)<br />
#* Press "Export Data..." In the dialog that comes up, select "MATLAB Workspace" in the "Data Destination" popup and type in a variable name, such as <tt>AirForce14lp10x</tt>. Data from the image you acquired will be available in the Matlab workspace.<br />
#* Switch to the MATLAB command window and type <tt>whos AirForce14lp10x</tt>. The image is represented as a 492x656 matrix of 16-bit integers.<br />
#* To display the image, use the <tt>imshow</tt> command. <br />
#** When the 12-bit numbers from the camera get transferred to the computer, they are converted to 16-bit numbers. 16-bit numbers can represent a range of values from 0-65535. This leaves a considerable portion of the number range unoccupied. Because of this, if you type <tt>imshow( AirForce14lp10x )</tt>, you will see an image that looks almost completely black. Adjust the image to fill the full range by typing <tt>imshow( 16.0037 * AirForce14lp10x )</tt>. 16.0037 equals 65535/4095. This factor maps values in the range 0-4095 to 0-65535.<br />
#* An even better way to work with images in MATLAB is to convert them to [[http://en.wikipedia.org/wiki/Double-precision_floating-point_format double precision floating point format]] straightaway. Double precision floating point numbers can represent an extremely wide range of values with high precision. Matlab includes a function for converting images, <tt>im2double</tt>. Type <tt>AirForce14lp10x = im2double( 16.0037 * AirForce14lp10x );</tt> to make the conversion and then use <tt>imshow</tt> to see the result. After the conversion to double, the range of intensities is mapped to 0-1, with 1 being full intensity and 0 completely dark.<br />
#* Use the data cursor to measure magnification<br />
#** When choosing the size of lines to image, consider the factors that influence the uncertainty of your measurement.<br />
#* Save your image as a .mat for later use in MATLAB (<tt>save AirForce14lp10x</tt>) or as a PNG image for use in your report or other programs. if you converted the image to double, the command might look something like: <tt>imwrite( im2uint16( AirForce14lp10x ), 'AirForce14lp10x.png', 'png' );</tt>.<br />
# Repeat the magnification measurement for the 40x and 100x objectives. <br />
#* With the 100x objective, you may want to substitute the Air Force target with a Ronchi Ruling, a grating with 600 line pairs per millimeter.<br />
# Calculate the FOV of the microscope using all three objectives.<br />
<br />
==Particle size measurement==<br />
[[Image:20.309_130813_BF_3p2umbeads_40x.png|right|thumb|Example image of 3.2 μm beads using the instructor microscope. Submit picture to replace this!]]<br />
<br />
Now that you know the magnification of your instrument, use it to measure the size of some microscopic objects as imaged with the 40x objective lens only. Slides with 7.2 μm, 3.2 μm and 1 μm silica microspheres are available in the lab.<br />
<br />
# Image 7.2 μm, 3.2 μm and 1 μm silica microspheres as described in the magnification measurement procedure (40x objective only). <br />
# Measure and report the average size and uncertainty of the spheres in each sample. How many spheres should you measure?<br />
<br />
==Microscope storage==<br />
<br />
During the microscopy lab, approximately seven thousand optical components will be taken from stock, assembled into microscopes, and properly returned to their assigned places. Please observe the following:<br />
* Store your microscope in one of the cubby holes in 16-336 (not in the lab). If you use one of the high shelves, get somebody to help you lift.<br />
* Keep all of the boxes for the optics you use with your instrument to simplify putting things away. <br />
* Take a blue bin to store loose items (such as lens boxes) in.<br />
* Stages, CCD cameras, neutral density filters and barrier filters stay at the lab station. Do not store these with your microscope.<br />
* Return objective lenses to the drawer when you are not using them. (Do not store them with your microscope.)<br />
* The stages are very expensive. Always lift from the bottom.<br />
* If you break something (or discover something pre-broken for you), do not return it to the component stock. Give all broken items to an instructor. You will not be penalized for breaking something, but not reporting may be looked upon less kindly.<br />
<br />
==Report outline==<br />
<br />
Find and follow all guidelines on the [[Microscopy report outline]] wiki page. <br />
<br />
{{:Optical Microscopy: Part 1 Report Outline}}<br />
<br />
{{:Optical microscopy lab wiki pages}}<br />
<br />
==References==<br />
<references /><br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/20.309:Course_Information20.309:Course Information2015-09-19T19:48:04Z<p>Steven Nagle: /* Lab hours and scheduling */</p>
<hr />
<div>[[Category:20.309]]<br />
[[Category:Needs review]]<br />
{{Template:20.309}}<br />
<br />
==Course meetings==<br />
<br />
Lecture: T/R/F 12:00n-1:00pm (32-155) <br />
<br />
Lab: open scheduling, see below (16-352)<br />
<br />
==Lab hours and scheduling==<br />
<br />
Use the [http://measurebiology.org/reservations/Web/? lab station reservation system] to reserve a place in the lab. (Be careful to reserve a time when the lab is actually open, since the scheduler will allow you to reserve a station outside of lab open hours.) Most lab exercises can be completed in about 6 hours per week; however, some students find the lab work significantly more or less time consuming. If you have little experience building things, you might want to plan for a few extra hours.<br />
<br />
'''New hours for Spring 2014'''<br />
{| border = "1" align="center" style="text-align:center"<br />
! width="80" | Monday<br />
!! width="80" | Tuesday<br />
!! width="80" | Wednesday<br />
!! width="80" | Thursday<br />
!! width="80" | Friday<br />
!! width="80" | Saturday<br />
!! width="80" | Sunday<br />
|-<br />
|1-6PM||<br />
{| align="center"<br />
|1-9PM<br />
|}<br />
||1-9PM||<br />
{| align="center"<br />
|1-7PM<br />
|}<br />
||1-6PM||Closed||1-6PM<br />
|}<br />
<br />
Monday and Wednesday mornings are "by appointment" starting at 11 am. To request early hours on Monday, reserve a station and [mailto:scwass@mit.edu email scwass@mit.edu] at least 48 hours in advance. To request early hours on Wednesday, reserve a station and [mailto:sfnagle@mit.edu email sfnagle@mit.edu] at least 48 hours in advance.<br />
<br />
During regular lab hours, please sign up at least 24 hours before coming to the lab. If there are no scholars signed up a day in advance, the lab may be closed without notice.<br />
<br />
The lab is located in room 16-352.<br />
<br />
Lab attendance is mandatory and there will be no make-up labs.<br />
<br />
==Safety==<br />
<br />
The chief hazards present in the 20.309 Lab come from laser radiation, chemical and biological materials, and electric equipment. Some simple precautions will make your time in the lab much safer.<br />
<br />
Get to know the [[20.309:Safety|20.309 Safety Page]]. Read the safety precautions in each lab manual.<br />
<br />
==Overview of laboratory modules==<br />
<br />
===Optics===<br />
<!--<br />
[[Image:Fluorescence Microscope Picture|right|150px|Fluorescence Microscope]]<br />
--><br />
'''''Fluorescence microscopy, particle tracking, and image processing'''''<br />
<br />
The first third of the semester is devoted to optical microscopy and imaging. Each student group will design and construct a microscope with trans-illuminated brightfield and epifluorescence imaging contrast modes. Students will characterize the resolution and mechanical stability of their microscopes, image a variety of samples, and make quantitative microrheological measurements by particle tracking.<br />
<br />
===Electronics===<br />
<!--<br />
[[Image:DNA Melting Apparatus Picture|right|150px|DNA Melting Apparatus]]<br />
--><br />
'''''Resistive networks, filters, and op-amp circuits for measurement'''''<br />
<br />
The second part of the course focuses on electronics. Over a series of labs, we will build several types of commonly used electronic circuits and combine them implement a system for measuring DNA melting curves. This section will also provide an introduction to computer control and data acquisition, including LabVIEW and MATLAB software.<br />
<br />
===Ultimate limits for force and position detection=== <br />
<br />
'''''Microcantilevers, precision measurement, and thermomechanical noise'''''<br />
<br />
Force sensors such as the optical tweezers and atomic force microscope (AFM) provide a unique means for investigating single biomolecules. Examples include the real-time monitoring of enzymatic activity with the optical tweezers and the direct measurement of forces required to unfold individual protein domains with the AFM. At the heart of these force sensors is an ultrasensitive displacement detector that resolves the position of compliant structure (i.e. microcantilever or optically trapped mircobead) with nanometer, or in some cases, sub-nanometer resolution. The performance of the force sensor is determined by the mechanical properties of the structure (spring constant, resonant frequency, damping, etc) and the resolution of the displacement detector. In this lab, we will measure the thermomechanical motion of a microcantilever sensor, estimate its detection limit, and compare to theoretical calculations. <br />
<br />
==Lab facilities==<br />
<br />
===Lab stations===<br />
<br />
There are 12 lab stations in room 16-352. Each station is equipped with:<br />
<br />
#anti-vibration optical table<br />
#digital oscilloscope<br />
#triple output power supply<br />
#tools in drawers<br />
#computer workstation with data acquisition card (DAQ)<br />
<br />
The lab also has several function generators.<br />
<br />
Stations 3, 6 and 11 are reserved for instructor use.<br />
<!-- TODO: add links to equipment manuals --><br />
<br />
===Computers===<br />
<br />
Lab stations are equipped with PCs running Windows 7. Each PC has a National Instruments data acquisition system. MatLab and LabVIEW software are installed on all lab PCs.<br />
<br />
Log in to the PCs as user <code>Learning Biologic Genie</code>. Ask an instructor for the password.<br />
<br />
Feel free to use the <code>Documents</code> folder while working at the PC, but beware that any data on the PCs could be erased without notice! At the end of your session transfer every file that you care about to your own computer or to a network location of your choice. The lab provides a network share named <code>Student Data</code>. You may access it via the link on the PC desktop or via the address <code>\\win.mit.edu\dfs\departmental\bioeng\bioenglab\StudentData</code>. Log in using your kerberos credentials.<br />
<br />
Additional course material can be found on our <code>Course Materials</code> share. There is also a link to this network share on the desktop, or you may the address <code>\\win.mit.edu\dfs\departmental\bioeng\bioenglab\CourseMaterials</code>. As before, log in using your kerberos credentials.<br />
<br />
===Course locker===<br />
<br />
The [http://web.mit.edu/~20.309 20.309 course locker] and Stellar site contain virtually every computer file you will need for the course. To access the locker on an Athena workstation, type <code>attach 20.309</code> and then <code>cd /mit/20.309</code>. Use the Desktop or Start Menu shortcut to access the locker from any PC in the lab. You can also access the locker with [http://itinfo.mit.edu/product.php?name=securefx&platform=Windows SecureFX].<br />
<br />
==Grading==<br />
{| class="wikitable" style="margin: 1em auto 1em auto;"<br />
|-<br />
|2 group lab reports||25%<br />
|-<br />
|2 individual oral presentations||25%<br />
|-<br />
|3 exams||30%<br />
|-<br />
|2 mini-labs||5%<br />
|-<br />
|class participation||10%<br />
|-<br />
|8 problem sets||5%<br />
|}<br />
<br />
==Lectures==<br />
<br />
Three lectures per week introduce key concepts behind the labs. An underlying theme throughout all the lectures will be on signals analysis (e.g. Fourier transforms, power spectral density, convolution theorem, etc.) as applied to electrical, mechanical and optical systems.<br />
<br />
In some cases, the lectures will be closely related to ongoing lab modules, and in other cases, the lectures will develop material that will be used in a future lab module.<br />
<br />
==Assignments==<br />
<br />
All assignments should be turned in electronically via the course Stellar site. Late work will not be accepted without an excuse from the Dean's office. Check the schedule on Stellar for assignment due dates and times. Your lowest homework grade will be dropped. Use your freebie to accommodate special situations such as interview travel.<br />
<br />
You are encouraged to seek advice from the instructors, TAs and other students; however, the work that is turned in must be your own. <br />
===Lab assignment===<br />
Two laboratory modules require a written report: optical microscopy and DNA Melting. Reports for the two short lab exercises will be answer-book style.<br />
<br />
===Homework===<br />
Problem sets will help solidify concepts presented in lecture and also to prepare for upcoming labs and exams.<br />
<br />
</div></div>Steven Naglehttp://measurebiology.org/wiki/Optical_aberrationsOptical aberrations2015-09-16T18:44:25Z<p>Steven Nagle: </p>
<hr />
<div>{{Template:20.309}}<br />
==Optical aberrations==<br />
<br />
Considerations in choosing a lens<br />
<br />
In practice, as the polychromatic nature of white light is taken into consideration, and as imaging conditions depart from the Gaussian optics approximations that have framed our representations so far, optical aberrations get introduced into images formed by lenses. <br />
Aberrations fall into two main categories: aberrations caused by wavelength variations (chromatic), and aberrations caused by the lens's spherical construction (these are known as Seidel's aberrations: spherical, coma, astigmatism, field curvature, and distortion) <ref>[http://micro.magnet.fsu.edu/primer/anatomy/aberrationhome.html http://micro.magnet.fsu.edu/primer/anatomy/aberrationhome.html]</ref>.<br />
<br />
===Chromatic aberration===<br />
[[Image:20.309 130822 ChromaticAberration.png|thumb|right|400 px|From fsu.edu <ref>[http://micro.magnet.fsu.edu/primer/anatomy/aberrations.html http://micro.magnet.fsu.edu/primer/anatomy/aberrations.html]</ref> Axial chromatic aberration and achromat doublet correction]]<br />
<br />
* Axial chromatic aberration is due to dispersion by the lens's medium whose index of refraction effectively varies with the light wavelength: the blue part of the spectrum is refracted to a greater extent than the red portion.<br />
* Lateral chromatic aberration manifests itself by a difference in magnification of blue ''vs.'' red images of white-light-illuminated objects, which causes color ringing at the outer regions of the field of view.<br />
* An achromatic doublet combination (the association of a converging lens with a weaker diverging lens) can correct chromatic aberrations for certain wavelengths.<br />
<br />
===Spherical aberration===<br />
[[Image:20.309 130822 SphericalAberration.png|thumb|right|300 px|Longitudinal and transverse spherical aberrations]]<br />
<br />
* Peripheral rays and axial rays have different focal points, because the former are actually refracted to a greater degree than the latter.<br />
* Spherical aberrations arise from the higher-than-first-order terms in the ''sin &theta;'' and ''cos &theta;'' expansions that become non-negligible as the incident light angle ''&theta;'' increases.<br />
* Spherical aberration causes the image to appear hazy or blurred and slightly out of focus.<br />
* This effect significantly degrades the resolution of the lens because it affects the coincident imaging of points ''on'' and ''off'' the optical axis.<br />
<br />
<br /><br />
===Coma and astigmatism===<br />
[[Image:20.309 130828 Astigmatism.png|thumb|right|300 px|In coma and astigmatism, parallel off-axis ''oblique'' rays do not focus at one point in the image plane, but rather at distinct points in the sagittal and meridional planes, causing image distortion.]]<br />
<br />
* Comatic aberration results in off-axis point objects appearing asymmetrical and taking a comet-like shape. Comatic aberration is most commonly encountered when a microscope is out of alignment.<br />
* Astigmatism engender ellipses or blurred lines as images of speciment points. Depending on the angle of the off-axis rays entering the lens, the line image may be oriented either tangentially or radially.<br />
<br />
<br /><br />
<br /><br />
<br /><br />
<br /><br />
<br /><br />
<br /><br />
<br />
===Petzval distortion or field curvature===<br />
[[Image:20.309 130826 PetzvalDistortion2.png|thumb|right|300 px|Petzval distortion results in image curvature.]]<br />
<br />
* Instead of generating image points of a flat object onto a flat screen as we have idealized so far in ray tracing estimations, a simple lens focuses these image points onto a spherical surface, shaped as a curved bowl whose curvature, the Petzval curvature, is the reciprocal of the lens radius.<br />
<br />
<br /><br />
<br /><br />
<br /><br />
<br /><br />
<br /><br />
<br /><br />
==References==<br />
<references /><br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/Optical_aberrationsOptical aberrations2015-09-16T18:36:42Z<p>Steven Nagle: /* Petzval distortion or field curvature */</p>
<hr />
<div>{{Template:20.309}}<br />
==Optical aberrations==<br />
<br />
Considerations in choosing a lens<br />
<br />
In practice, as the polychromatic nature of white light is taken into consideration, and as imaging conditions depart from the Gaussian optics approximations that have framed our representations so far, optical aberrations get introduced into images formed by lenses. <br />
Aberrations fall into two main categories: aberrations caused by wavelength variations (chromatic), and aberrations caused by the lens's spherical construction (these are known as Seidel's aberrations: spherical, coma, astigmatism, field curvature, and distortion) <ref>[http://micro.magnet.fsu.edu/primer/anatomy/aberrationhome.html http://micro.magnet.fsu.edu/primer/anatomy/aberrationhome.html]</ref>.<br />
<br />
[[Image:20.309 130822 ChromaticAberration.png|thumb|right|400 px|From fsu.edu <ref>[http://micro.magnet.fsu.edu/primer/anatomy/aberrations.html http://micro.magnet.fsu.edu/primer/anatomy/aberrations.html]</ref> Axial chromatic aberration and achromat doublet correction]]<br />
===Chromatic aberration===<br />
<br />
* Axial chromatic aberration is due to dispersion by the lens's medium whose index of refraction effectively varies with the light wavelength: the blue part of the spectrum is refracted to a greater extent than the red portion.<br />
* Lateral chromatic aberration manifests itself by a difference in magnification of blue ''vs.'' red images of white-light-illuminated objects, which causes color ringing at the outer regions of the field of view.<br />
* An achromatic doublet combination (the association of a converging lens with a weaker diverging lens) can correct chromatic aberrations for certain wavelengths.<br />
<br />
[[Image:20.309 130822 SphericalAberration.png|thumb|right|300 px|Longitudinal and transverse spherical aberrations]]<br />
===Spherical aberration===<br />
<br />
* Peripheral rays and axial rays have different focal points, because the former are actually refracted to a greater degree than the latter.<br />
* Spherical aberrations arise from the higher-than-first-order terms in the ''sin &theta;'' and ''cos &theta;'' expansions that become non-negligible as the incident light angle ''&theta;'' increases.<br />
* Spherical aberration causes the image to appear hazy or blurred and slightly out of focus.<br />
* This effect significantly degrades the resolution of the lens because it affects the coincident imaging of points ''on'' and ''off'' the optical axis.<br />
<br />
===Coma and astigmatism===<br />
<br />
* Comatic aberration results in off-axis point objects appearing asymmetrical and taking a comet-like shape. Comatic aberration is most commonly encountered when a microscope is out of alignment.<br />
* Astigmatism engender ellipses or blurred lines as images of speciment points. Depending on the angle of the off-axis rays entering the lens, the line image may be oriented either tangentially or radially.<br />
<br />
[[Image:20.309 130828 Astigmatism.png|thumb|right|300 px|In coma and astigmatism, parallel off-axis ''oblique'' rays do not focus at one point in the image plane, but rather at distinct points in the sagittal and meridional planes, causing image distortion.]]<br />
<br />
===Petzval distortion or field curvature===<br />
[[Image:20.309 130826 PetzvalDistortion2.png|thumb|right|300 px|Petzval distortion results in image curvature.]]<br />
<br />
* Instead of generating image points of a flat object onto a flat screen as we have idealized so far in ray tracing estimations, a simple lens focuses these image points onto a spherical surface, shaped as a curved bowl whose curvature, the Petzval curvature, is the reciprocal of the lens radius.<br />
<br />
==References==<br />
<references /><br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/Optical_aberrationsOptical aberrations2015-09-16T18:36:30Z<p>Steven Nagle: /* Coma and astigmatism */</p>
<hr />
<div>{{Template:20.309}}<br />
==Optical aberrations==<br />
<br />
Considerations in choosing a lens<br />
<br />
In practice, as the polychromatic nature of white light is taken into consideration, and as imaging conditions depart from the Gaussian optics approximations that have framed our representations so far, optical aberrations get introduced into images formed by lenses. <br />
Aberrations fall into two main categories: aberrations caused by wavelength variations (chromatic), and aberrations caused by the lens's spherical construction (these are known as Seidel's aberrations: spherical, coma, astigmatism, field curvature, and distortion) <ref>[http://micro.magnet.fsu.edu/primer/anatomy/aberrationhome.html http://micro.magnet.fsu.edu/primer/anatomy/aberrationhome.html]</ref>.<br />
<br />
[[Image:20.309 130822 ChromaticAberration.png|thumb|right|400 px|From fsu.edu <ref>[http://micro.magnet.fsu.edu/primer/anatomy/aberrations.html http://micro.magnet.fsu.edu/primer/anatomy/aberrations.html]</ref> Axial chromatic aberration and achromat doublet correction]]<br />
===Chromatic aberration===<br />
<br />
* Axial chromatic aberration is due to dispersion by the lens's medium whose index of refraction effectively varies with the light wavelength: the blue part of the spectrum is refracted to a greater extent than the red portion.<br />
* Lateral chromatic aberration manifests itself by a difference in magnification of blue ''vs.'' red images of white-light-illuminated objects, which causes color ringing at the outer regions of the field of view.<br />
* An achromatic doublet combination (the association of a converging lens with a weaker diverging lens) can correct chromatic aberrations for certain wavelengths.<br />
<br />
[[Image:20.309 130822 SphericalAberration.png|thumb|right|300 px|Longitudinal and transverse spherical aberrations]]<br />
===Spherical aberration===<br />
<br />
* Peripheral rays and axial rays have different focal points, because the former are actually refracted to a greater degree than the latter.<br />
* Spherical aberrations arise from the higher-than-first-order terms in the ''sin &theta;'' and ''cos &theta;'' expansions that become non-negligible as the incident light angle ''&theta;'' increases.<br />
* Spherical aberration causes the image to appear hazy or blurred and slightly out of focus.<br />
* This effect significantly degrades the resolution of the lens because it affects the coincident imaging of points ''on'' and ''off'' the optical axis.<br />
<br />
===Coma and astigmatism===<br />
<br />
* Comatic aberration results in off-axis point objects appearing asymmetrical and taking a comet-like shape. Comatic aberration is most commonly encountered when a microscope is out of alignment.<br />
* Astigmatism engender ellipses or blurred lines as images of speciment points. Depending on the angle of the off-axis rays entering the lens, the line image may be oriented either tangentially or radially.<br />
<br />
[[Image:20.309 130828 Astigmatism.png|thumb|right|300 px|In coma and astigmatism, parallel off-axis ''oblique'' rays do not focus at one point in the image plane, but rather at distinct points in the sagittal and meridional planes, causing image distortion.]]<br />
<br />
===Petzval distortion or field curvature===<br />
<br />
* Instead of generating image points of a flat object onto a flat screen as we have idealized so far in ray tracing estimations, a simple lens focuses these image points onto a spherical surface, shaped as a curved bowl whose curvature, the Petzval curvature, is the reciprocal of the lens radius.<br />
<br />
==References==<br />
<references /><br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/Optical_aberrationsOptical aberrations2015-09-16T18:36:15Z<p>Steven Nagle: /* Spherical aberration */</p>
<hr />
<div>{{Template:20.309}}<br />
==Optical aberrations==<br />
<br />
Considerations in choosing a lens<br />
<br />
In practice, as the polychromatic nature of white light is taken into consideration, and as imaging conditions depart from the Gaussian optics approximations that have framed our representations so far, optical aberrations get introduced into images formed by lenses. <br />
Aberrations fall into two main categories: aberrations caused by wavelength variations (chromatic), and aberrations caused by the lens's spherical construction (these are known as Seidel's aberrations: spherical, coma, astigmatism, field curvature, and distortion) <ref>[http://micro.magnet.fsu.edu/primer/anatomy/aberrationhome.html http://micro.magnet.fsu.edu/primer/anatomy/aberrationhome.html]</ref>.<br />
<br />
[[Image:20.309 130822 ChromaticAberration.png|thumb|right|400 px|From fsu.edu <ref>[http://micro.magnet.fsu.edu/primer/anatomy/aberrations.html http://micro.magnet.fsu.edu/primer/anatomy/aberrations.html]</ref> Axial chromatic aberration and achromat doublet correction]]<br />
===Chromatic aberration===<br />
<br />
* Axial chromatic aberration is due to dispersion by the lens's medium whose index of refraction effectively varies with the light wavelength: the blue part of the spectrum is refracted to a greater extent than the red portion.<br />
* Lateral chromatic aberration manifests itself by a difference in magnification of blue ''vs.'' red images of white-light-illuminated objects, which causes color ringing at the outer regions of the field of view.<br />
* An achromatic doublet combination (the association of a converging lens with a weaker diverging lens) can correct chromatic aberrations for certain wavelengths.<br />
<br />
[[Image:20.309 130822 SphericalAberration.png|thumb|right|300 px|Longitudinal and transverse spherical aberrations]]<br />
===Spherical aberration===<br />
<br />
* Peripheral rays and axial rays have different focal points, because the former are actually refracted to a greater degree than the latter.<br />
* Spherical aberrations arise from the higher-than-first-order terms in the ''sin &theta;'' and ''cos &theta;'' expansions that become non-negligible as the incident light angle ''&theta;'' increases.<br />
* Spherical aberration causes the image to appear hazy or blurred and slightly out of focus.<br />
* This effect significantly degrades the resolution of the lens because it affects the coincident imaging of points ''on'' and ''off'' the optical axis.<br />
<br />
===Coma and astigmatism===<br />
<br />
* Comatic aberration results in off-axis point objects appearing asymmetrical and taking a comet-like shape. Comatic aberration is most commonly encountered when a microscope is out of alignment.<br />
* Astigmatism engender ellipses or blurred lines as images of speciment points. Depending on the angle of the off-axis rays entering the lens, the line image may be oriented either tangentially or radially.<br />
<br />
[[Image:20.309 130826 PetzvalDistortion2.png|thumb|right|300 px|Petzval distortion results in image curvature.]]<br />
===Petzval distortion or field curvature===<br />
<br />
* Instead of generating image points of a flat object onto a flat screen as we have idealized so far in ray tracing estimations, a simple lens focuses these image points onto a spherical surface, shaped as a curved bowl whose curvature, the Petzval curvature, is the reciprocal of the lens radius.<br />
<br />
==References==<br />
<references /><br />
<br />
{{Template:20.309 bottom}}</div>Steven Naglehttp://measurebiology.org/wiki/Optics_BootcampOptics Bootcamp2015-09-16T03:23:29Z<p>Steven Nagle: /* Visualize, capture, and save images in Matlab */</p>
<hr />
<div>[[Category:Lab Manuals]]<br />
[[Category:20.309]]<br />
[[Category:Optical Microscopy Lab]]<br />
{{Template:20.309}}<br />
<br />
[[Image: 140730_OpticsBootcamp_3.jpg|thumb|250px|right|Imaging apparatus with illuminator, object, lens, and CCD camera mounted on an optical rail.]]<br />
<br />
==Overview==<br />
This lab exercise will introduce you to some of the optical components that you will use in the microscopy lab. You will build an apparatus that includes an LED illuminator, an object with precisely spaced markings, a lens, and a CCD camera. You will place the object at several distances from the lens and measure the corresponding image distance. Using the MATLAB Image Acquisition Tool, you will record images and use them to measure magnification. Finally, you will compare your measurements to the values given by the lens makers' formula that was derived in class.<br />
<br />
Before you get started, take some time to learn your way around the lab. [[Lab orientation|This page]] gives an overview of all the wonderful resources in the lab.<br />
Now go ahead and build your lens measuring apparatus. <br />
<br />
==Gather materials==<br />
[[Image: 140729_OpticsBootcamp_01.jpg|thumb|right|Parts for the simple imaging apparatus.]] <br />
<br />
The first step is to gather the materials required to build the lens measuring apparatus. The lists below include part numbers and descriptive names of all the components in the apparatus. It is likely that you will find some of the terms not-all-that-self-explanatory. Most of the parts are manufactured by a company called ThorLabs. If you have a question about any of the components, the [http://www.thorlabs.com ThorLabs website] can be very helpful. For example, if the procedure calls for an SPW602 spanner wrench and you have no idea what such a thing might look like, try googling the term: "thorlabs SPW602". You will find your virtual self just a click or two away from [http://www.thorlabs.com/thorproduct.cfm?partnumber=SPW602 a handsome photo and detailed specifications].<br />
<br />
===Optomechanics===<br />
These components are located in plastic bins on top of the center parts cabinet:<br />
<br />
* 1 x RLA1800 dovetail optical rails<br />
* 4 x RC1 rail carriers<br />
* 1 x SM1L10 lens tube<br />
* 1 x SM1RC lens tube slip ring<br />
* 1 x CP02 cage plate<br />
* 1 x LCP01 cage plate (looks like an "O" in a square)<br />
* 1 x LCP02 cage plate adapter (looks like an "X")<br />
* 2 x SM2RR retaining rings<br />
<br />
These components are located on the counter above the west drawers.<br />
<br />
* 4 x ER1 cage assembly rod (The last digit of the part number is the length in inches. Take a 1" rod. Lengths less than 1" have a part number that starts with a zero.)<br />
* 6 x SM1RR retaining rings<br />
<br />
===Screws and posts===<br />
Stainless steel, &frac14;-20 size, socket head cap screws (SHCS), washers, posts, and post holders are located on top of the west parts cabinet. If you are unfamiliar with screw types, take a look at the main screw page on the [http://www.mcmaster.com/#screws/=tjwmu5 McMaster-Carr website]. Notice on the left side of the page that there are ''about ...'' links on the left side of the page. Click the links for more information about screw sizes and attributes. [http://www.boltdepot.com/fastener-information/printable-tools/Socket-Cap-Size-Chart.pdf This link] will take you to an awesome chart of SHCS sizes.<br />
<br />
* 4 x PH2 post holders<br />
* 4 x TR2 optical posts<br />
* 4 x 8-32 set screws<br />
* 6 x 1/4-20 x 5/16" socket cap screws<br />
* 1 x 1/4-20 set screw<br />
<br />
===Optics===<br />
Lenses and microscope objectives are located in the west drawers.<br />
<br />
* 1 x LA1951 plano-convex, f = 25 mm lens<br />
* 1 x LB1811 biconvex, f = 35 mm lens<br />
<br />
===Object===<br />
Imaging targets are located in a plastic bin on top of the east cabinet.<br />
<br />
* 1 x R1DS1N 1951 USAF test target<br />
<br />
===Optoelectronics===<br />
LEDs will be in a plastic bin on top of the center cabinet.<br />
<br />
* 1 x red, super-bright LED (mounted)<br />
<br />
===Tools===<br />
Most of the tools are located in the drawers at your lab station. Be sure to put all of the tools you use back in their proper location.<br />
<br />
Hex keys (also called Allen wrenches) are used to operate SHCSs. Some hex keys have a flat end and others have a ball on the end, called balldrivers. The ball makes it possible to use the driver at an angle to the screw axis, which is very useful in tight spaces. You can get things tighter (and tight things looser) with a flat driver.<br />
<br />
* 1 x 3/16 hex balldriver for 1/4-20 cap screws<br />
* 1 x 9/64 hex balldriver<br />
* 1 x 0.050" hex balldriver for 4-40 set screws (tiny)<br />
* 1 x SPW602 spanner wrench<br />
<br />
You will also need to use an adjustable spanner wrench. The adjustable spanner resides at the lens cleaning station. There is only one of these in the lab. It is likely that one of your classmates neglected to return it to the proper place. This situation can frequently be remedied by yelling, "who has the adjustable spanner wrench?" at the top of your lungs. Try not to use any expletives. And please return the adjustable spanner wrench to the lens cleaning station when you are done.<br />
<br />
* 1 x SPW801 adjustable spanner wrench<br />
<br />
===Things that should already be (and stay at) your lab station===<br />
* 1 x Manta CCD camera<br />
* 1 x Calrad 45-601 power adapter for CCD<br />
* 1 x ethernet cable connected to the lab station computer<br />
<br />
==Build the apparatus==<br />
<br />
{|class="wikitable"<br />
|width="150"|[[Image: 140729_OpticsBootcamp_03.jpg|frameless|150px]]<br />
|width="150"|[[Image: 140729_OpticsBootcamp_09.jpg|frameless|150px]]<br />
|align="left" colspan="2"|'''Optical rails'''<br />
Optical rails are useful for arranging components in a line that require variable separation. Sliding clamps sit on the rail. The clamps have a thumbscrew that locks them in position.<br />
* Secure the optical rail on the optical table using two 1/4-20 x 5/16 cap screws and the 3/16 hex balldriver.<br />
* Prepare four sliding posts, each by attaching one RC1 rail carrier to one PH2-ST post holder with one 1/4-20 x 5/16 cap screws.<br />
|-<br />
|width="150"|[[Image: 140729_OpticsBootcamp_05.jpg|frameless|150px]] <br />
|width="150"|[[Image: 140729_OpticsBootcamp_07.jpg|frameless|150px]] <br />
|align="left" rowspan = "3" colspan="2"|'''Mount the LED light source''':<br />
* In the LCP01 cage plate, the LED will get sandwiched in-between two SM2RR retaining rings. First screw in one SM2RR only 1 mm deep.<br />
* Next place the LED above it. <br />
* Finally tighten down the second SM2RR using the SPW801 adjustable spanner wrench. The SPW801 can be opened until its width matches the SM2RR diameter, the separation between the ring's notches.<br />
* In the LCP02 cage plate adapter, screw in on SM1RR 3 mm deep.<br />
* Carefully (use lens paper unsparingly to protect the lens surface) place the 25 mm plano-convex lens above it, with the hemisphere facing down ''yet not touching any potentially damaging surface''.<br />
* Tighten down a second SM1RR using the SPW602 spanner wrench, whose guide flanges sit in the ring's notches to prevent any scratching of the lens's optical surface.<br />
* Attach the LCP01 cage plate (holding the LED) to the LCP02 adapter (holding the 25 mm condenser lens), using the 0.050" hex balldriver to secure four ER1 rods with eight 4-40 set screws.<br />
* Affix a TR2 optical post to the LCP01 cage plate (holding the LED).<br />
* Slide in the ''LED'' assembly along the optical rail.<br />
'''Power the LED light source''':<br />
* The red LED will be connected to a DC power supply. Ensure that the current limit on the power supply (CH1) is set to a value below 0.5 A.<br />
* Connect channel CH1 to the red and black threads of the LED, using alligator clip cables.<br />
* Turn on the power supply, and press its ''Output'' button to light the LED.<br />
* Adjust the LED brightness using the power supply's ''Voltage'' knob.<br />
|-<br />
|width="150"|[[Image: 140729_OpticsBootcamp_10.jpg|frameless|150px]] <br />
|width="150"|[[Image: 140729_OpticsBootcamp_11.jpg|frameless|150px]] <br />
|-<br />
|width="150"|[[Image: 140729_OpticsBootcamp_12.jpg|frameless|150px]] <br />
|width="150"|[[Image: 140729_OpticsBootcamp_13.jpg|frameless|150px]] <br />
|-<br />
|width="150"|[[Image: 140729_OpticsBootcamp_08.jpg|frameless|120px]]<br />
|width="150"|[[Image: 140729_OpticsBootcamp_15.jpg|frameless|150px]]<br />
|width="200"|[[Image: 140730_OpticsBootcamp_1.jpg|frameless|200px]] <br />
|width="200"|[[Image: 140730_OpticsBootcamp_2.jpg|frameless|150px]] <br />
|-<br />
|width="150"|[[Image: 140730_OpticsBootcamp_4.jpg|frameless|150px]] <br />
|align="left" colspan="3" rowspan="2"|'''Mount the object (US Air Force target 1951)''':<br />
* Tighten the R1DS1N 1951 USAF test target in-between two SM1RR retaining rings inside the SM1L10 lens tube, using the SPW602 spanner wrench. (This procedure should be reminiscent of the insertion of the 25 mm hemispherical lens in the cage plate adapter.)<br />
* Slide in the lens tube through the SM1RC slip ring. By rotating the lens tube, you will be able to modify orientation of the ''object''.<br />
* Lock the lens tube in place using the 9/64 hex balldriver.<br />
* Affix a TR2 optical post to the SM1RC slip ring (holding the USAF target).<br />
* Slide in the ''object'' assembly along the optical rail.<br />
'''Mount the lens (f = 35 mm)''':<br />
* Tighten the LB1811 biconvex f = 35 mm lens in-between two SM1RR retaining rings inside the CP02 cage plate. <br />
* Affix a TR2 optical post to the CP02 cage plate (holding the lens).<br />
* Slide in the ''lens'' assembly along the optical rail.<br />
|-<br />
|width="150"|[[Image: 140729_OpticsBootcamp_16.jpg|frameless|150px]]<br />
|-<br />
|width="150"|[[Image: 140729_OpticsBootcamp_17.jpg|frameless|150px]]<br />
|width="150"|[[Image: 140729_OpticsBootcamp_18.jpg|frameless|150px]]<br />
|align="left" colspan="2"|'''Mount the CCD camera''': <br />
* Affix a TR2 optical post directly to the CCD camera plate using the 1/4-20 set screw.<br />
* Slide in the ''camera'' assembly along the optical rail.<br />
* Connect the CCD to the computer using a red ethernet cable.<br />
* Power up the CCD using the Calrad 45-601 power adapter.<br />
|-<br />
|align="left" colspan="4"|<br />
<br />
'''Vertically align the ''LED'', ''object'', ''lens'', and ''camera'' assemblies.'''<br />
* Make sure the heights of the components are adjusted so your light is going through the object and the lens.<br />
* Also check that the camera height and angle are appropriately positioned to receive the image of the object.<br />
|}<br />
<br />
==Visualize, capture, and save images in Matlab==<br />
<br />
[[Image: 20.309 130909 ImagingWithLens.png|right|thumb|300px]]<br />
<br />
Now that you've learned the basics of mounting, aligning and adjusting optical components, you will through this lab exercise<br />
* verify the lens maker and the magnification formulae: <br />
:<math> {1 \over S_o} + {1 \over S_i} = {1 \over f}</math><br />
:<math>M = {h_i \over h_o} = {S_i \over S_o}</math><br />
* become familiar with image acquisition and distance measurement using the Matlab software.<br />
<br />
{|class="wikitable"<br />
|width="250"|[[Image: 140730_Matlab_01.png|frameless|250px]]<br />
|align="left"|<br />
* Log on to the computer, launch Matlab, and run <tt>imaqtool</tt>.<br />
* Select the ''Manta_G-032B(gentl-1) Mono 12" hardware in the left bar.<br />
* The ''Start Preview'' button will bring up a window of the live image from the CCD camera.<br />
* Move the lens and USAF 1951 target object to produce a focused image.<br />
** Start with the USAF 1951 target at the 2<math>f</math> position, i.e., 70 mm from the lens.<br />
** Divide additional images into an equal number with the target, the object, placed at less than and greater than 2<math>f</math> from the lens<br />
* Under the ''Device Properties'' tab, optimize the ''Exposure Time Abs'' field for good contrast without pixel saturation.<br />
* Measure the distance <math>S_o</math> from the target object to the lens and the distance <math>S_i</math> from the lens to the CCD active imaging plane.<br />
** Does the lens maker formula <math> {1 \over S_o} + {1 \over S_i} = {1 \over f}</math> apply as it should when the image focus is optimized?<br />
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|width="250"|[[Image: 140730_Matlab_02.png|frameless|250px]]<br />
|align="left"|<br />
* Save images in Matlab:<br />
** Make sure you limit to 1 the number of ''Frames Per Trigger'' in the ''General'' tab of the ''Acquisition Parameters'';<br />
** Use the ''Start Acquisition'' and ''Export Data'' buttons;<br />
** Navigate to the ''CourseMaterials\StudentData\Fall 2015\'' directory accessible from the computer desktop to save your data files remotely on a server you'll be able to browse from your home computer.<br />
*** Recall from one of the initial Stellar announcements that you must use your kerberos ID, preceded by win\, as your username. For example, Professor Nagle would enter "win\sfnagle". Use your kerberos password as well. Remember to disconnect the mapped drive when you are done at your lab station, or log out of the Windows session entirely.<br />
** The file extension will be .MAT (''e.g.'' <tt>1951target_01.mat</tt>), although this extension will not be visible in the Windows Explorer. The variable within this file (''e.g.'' <tt>im01</tt>) will represent the image as a 492x656 matrix of 16-bit integers. <br />
|}<br />
<br />
==Examine images in Matlab==<br />
{|class="wikitable"<br />
|width="250"|[[Image: 140730_Matlab_03.png|frameless|250px]]<br />
|align="left"|<br />
* To display the image in Matlab, use the <tt>imshow</tt> command:<br />
** In Matlab, ''open'' your saved image file ('1951target_01.mat') from the ''Student Data\ Fall 2015\'' directory.<br />
** Its contents 'im01' now appear in your workspace.<br />
** When the 12-bit numbers from the camera get transferred to the computer, they are converted to 16-bit numbers. 16-bit numbers can represent a range of values from 0-65535. This leaves a considerable portion of the number range unoccupied. Because of this, if you type <tt>imshow( im01 )</tt>, you will see an image that looks almost completely black. <br />
** Adjust the image to fill the full range by typing <tt>imshow( 16.0037 * im01 )</tt>. <br />
::''Note:'' 16.0037 equals 65535 / 4095. This factor maps values in the range 0-4095 to 0-65535.<br />
|-<br />
|width="250"|[[Image: 140730_Matlab_04.png|frameless|250px]]<br />
|align="left" rowspan="2"|<br />
* Determine the distance (in pixels) between two specific points in the image:<br />
** Either use the ''Data Cursor Tool'' and some trigonometry to display the X and Y coordinate of your mouse pointer;<br />
** or type <code>imdistline</code> on the console to make a very useful measuring tool appear on the image (recommended);<br />
** or use the interactive <tt>improfile</tt> function from the Matlab command window, which lets you trace a segment across the active figure (visualized as a dotted line) and generates a plot of pixel intensity vs. pixel position along the segment in a new figure.<br />
** This manipulation allows you to calculate the image size <math>h_i</math>, taking into account the CCD pixel size: 7.4 &mu;m x 7.4 &mu;m.<br />
* Confirm the corresponding object size <math>h_o</math>: <br />
** Refer to the [https://en.wikipedia.org/wiki/1951_USAF_resolution_test_chart specification sheet of the USAF 1951 test target], pages 5 and 8 in particular.<br />
** The 1" R1DS1N 1951 USAF test target includes elements of groups 2 and 3.<br />
** Example: ''Element 2'' in ''Group 2'' has 4.49 cycles (= line pairs) per millimeter. So 2 line pairs from ''Element 2'' in ''Group 2'' span 2 &divide; 4.49 = 0.4454 mm = 445.4 &mu;m.<br />
* Do both magnification relationships <math>M = {h_i \over h_o} = {S_i \over S_o}</math> match ?<br />
|-<br />
|width="250"|[[Image: 140730_Matlab_05.png|frameless|250px]]<br />
|}<br />
<br />
==Plot and discuss your results==<br />
* Repeat these measurements of <math>S_o</math>, <math>S_i</math>, <math>h_o</math>, and <math>h_i</math> for several values of <math>S_o</math>.<br />
* Plot <math>{1 \over S_i}</math> as a function of <math> {1 \over f} - {1 \over S_o}</math>.<br />
* Plot <math>{h_i \over h_o}</math> as a function of <math>{S_i \over S_o}</math>.<br />
* What sources of error affect your measurements?<br />
* Given the sources of error, how far off could your measurements of magnification be?<br />
<br />
Once you are done with your measurements, please clean up and put back all the parts.<br />
<br />
{{:Optical microscopy lab wiki pages}}<br />
<br />
{{Template:20.309 bottom}}</div>Steven Nagle