Difference between revisions of "Assignment 1 Overview: Transillumination microscopy"

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(Lab exercise 2: imaging with a lens)
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[[Category:Optical Microscopy Lab]]
 
[[Category:Optical Microscopy Lab]]
 
{{Template:20.309}}
 
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[[File:Mens et Manus.jpg|center|350 px]]
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''[https://www.youtube.com/watch?v=O7oD_oX-Gio You got your ''mens'' in my ''manus.]''
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<blockquote>
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''&mdash;[http://latin-dictionary.net/search/latin/mens Manus]''
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</blockquote>
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</div>
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</blockquote>
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<blockquote>
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''[https://www.youtube.com/watch?v=GuENAWds5B0 You got your ''manus'' in my ''mens.]''
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<blockquote>
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''&mdash;[http://latin-dictionary.net/search/latin/manus Mens]''
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</blockquote>
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</center>
  
 
==Assignment Details ==
 
==Assignment Details ==
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==Lab Instructions==
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==Lab Instructions, Part 1: getting oriented==
  
 
===Lab Orientation ===
 
===Lab Orientation ===
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{{:Lab orientation}}
 
{{:Lab orientation}}
  
===Lab exercise 1: measure focal length of lenses===
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===Exercise 1: measure focal length of lenses===
 +
 
 
Make your way to the lens measuring station. Measure the focal length of the four lenses marked A, B, C, and D located nearby.
 
Make your way to the lens measuring station. Measure the focal length of the four lenses marked A, B, C, and D located nearby.
  
===Lab exercise 2: imaging with a lens===
+
===Exercise 2: imaging with a lens===
  
 
<figure id="fig:Optics_bootcamp_apparatus">
 
<figure id="fig:Optics_bootcamp_apparatus">
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* What sources of error affect your measurements?
 
* What sources of error affect your measurements?
  
===Lab exercise 3: noise in images===
+
===Exercise 3: noise in images===
 
Almost all measurements of the physical world suffer from some kind of noise. Capturing an image involves measuring light intensity at numerous locations in space. The [https://www.alliedvision.com/en/products/cameras/detail/Manta/G-032.html Manta G-032] CCD cameras in the lab measure about 320,000 unique locations for each image. Every one of those measurements is subject to noise. In this part of the lab, you will quantify the random noise in images made with the Manta cameras, which are the same ones that you will use in the microscopy lab.  
 
Almost all measurements of the physical world suffer from some kind of noise. Capturing an image involves measuring light intensity at numerous locations in space. The [https://www.alliedvision.com/en/products/cameras/detail/Manta/G-032.html Manta G-032] CCD cameras in the lab measure about 320,000 unique locations for each image. Every one of those measurements is subject to noise. In this part of the lab, you will quantify the random noise in images made with the Manta cameras, which are the same ones that you will use in the microscopy lab.  
  
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(Need a refresher on loglog plots? [[Understanding log plots|Click here]].)
 
(Need a refresher on loglog plots? [[Understanding log plots|Click here]].)
  
=== Lab exercise 4: build a transillumination microscope
+
==Lab Instructions, Part 2: building a microscope==
  
 
<blockquote>
 
<blockquote>
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</blockquote>
 
</blockquote>
  
====Background materials and references====  
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===Background materials and references===
  
 
In this part of the lab you will jump right in to building a full-fledged microscope. The following online materials provide useful background.
 
In this part of the lab you will jump right in to building a full-fledged microscope. The following online materials provide useful background.
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** [http://www.microscopyu.com/articles/formulas/formulasresolution.html Resolution]
 
** [http://www.microscopyu.com/articles/formulas/formulasresolution.html Resolution]
  
===Microscope block diagram===
+
===Exercise 1: Assemble the microscope===
Before you begin building, you will draw out your microscope [[#Design|design]]. To get you started, here is an example block diagram of a 20.309 microscope. Note that for Part 1, you will be building the brightfield path. You should leave space for the fluorescence path, which you will complete in Part 2.
+
 
 +
====Microscope block diagram====
 +
To get you started, here is a block diagram of a 20.309 microscope. Note that for Assignment 1, you will be building the brightfieqld (or transillumination) path. You should leave space for the fluorescence path, which you will complete next week.
  
 
[[Image:20.309 130911 YourMicroscope.png|center|thumb|400px|20.309 microscope block diagram]]
 
[[Image:20.309 130911 YourMicroscope.png|center|thumb|400px|20.309 microscope block diagram]]
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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.
 
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.
 
===Microscope construction===
 
  
 
====Design====
 
====Design====
  
Sketch out the design for your microscope on paper. For this part of the lab, you can just draw the bright field illumination path. Include all the optical elements (lenses, mirrors, microscope objectives, and camera). Label all distances, lens specifications, and orientations. You will include this diagram (or a cleaned-up version) in your lab report.
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Sketch out the design for your microscope on paper (or print out the above diagram). For this part of the lab, you can just draw the bright field illumination path. Label all the optical elements (lenses, mirrors, microscope objectives, and camera), distances, lens specifications, and orientations.  
 
* 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×)?
 
* 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×)?
 
* Which sections of the light path can be open (strut-based structure, cage rods)? Which would better enclosed (Thorlabs lens tubes)?
 
* Which sections of the light path can be open (strut-based structure, cage rods)? Which would better enclosed (Thorlabs lens tubes)?
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* Which lens will you use between the LED and the sample for bright field transmitted light imaging?
 
* Which lens will you use between the LED and the sample for bright field transmitted light imaging?
  
====Practice====
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Next, take a look at the example microscope in the lab. This microscope is here for your reference, but please do not touch, alter, or remove parts from the example microscope.
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[[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]]
  
* 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.
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Can you identify all the components of your diagram? Try to think about the purpose of each component, and why it is laid out a particular way. Once you're satisfied that you understand the big picture, it's time to build your own!
* You may view the example microscope for reference. However, please do not touch, alter, or remove parts from the example microscope.
+
 
 +
A few tips to keep in mind:
 +
* Reproduce the general layout of the example microscope: it grants compactness and allows your device to be a stand-alone breadboard-transportable microscope.  
 
* 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:
 
* 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:
** Reproduce the general layout of the example microscope: it grants compactness and allows your device to be a stand-alone breadboard-transportable microscope.  
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 +
====Assemble the base====
 +
Gather the following parts:
 +
<gallery widths=216px caption="Optical breadboard(Located in the lower left cubby of the Instructor Cubbies):">
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File:OpticalBreadBoard.jpg|Optical breadboard
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File:VerticalMountingPost.jpg|Vertical Thorlabs P14 mounting post (1.5" diameter)
 +
</gallery>
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 +
* On a 1' x 2' x <sup>1</sup>/<sub>2</sub>" optical breadboard, 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 above picture is in error as the P14 is only 9 positions from a short side of the breadboard.)
 +
 
 +
====Assemble the illuminator====
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Gather parts:
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<gallery widths=216px caption="Optomechanics and LED (located in plastic bins on top of the center parts cabinet):">
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File:LensTube05.jpg|1 x 0.5" Lens tube (SM1L05)
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File:LCP01.jpg|1 x 2" Cage plate (LCP01, looks like an "O" in a square)
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File:LCP02.jpg|1 x Cage plate adapter (LCP02, looks like an "X")
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File:SM2RR.jpg|2 x 2" Retaining rings (SM2RR)
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File:RedLED.jpg|1 x red, super-bright LED (mounted in heatsink)
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</gallery>
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<gallery widths=216px caption="Optomechanics (located on the counter above the west drawers):">
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File:ER8.jpg|3 x ER2 cage assembly rod (The last digit of the part number is the length in inches.)
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File:SM1RR.jpg|1 x 1" Retaining rings (SM1RR)
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</gallery>
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<gallery widths=216px caption="Optics (located in the west drawers):">
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File:Lenses.jpg|1 x LA1951 plano-convex, f = 25 mm lens (this will be used as a condenser for your illuminator)
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</gallery>
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 +
Most of the tools you will need are located in the drawers next to your lab station. 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. Here is a list of the tools you will need:
 +
 
 +
<gallery widths=216px caption="Tools (located in your station's drawers):">
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File:BallDrivers.jpg|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)
 +
File:SPW602.jpg|1 x SPW602 spanner wrench
 +
</gallery>
 +
 
 +
You will also need to use an adjustable spanner wrench. The adjustable spanner resides at the lens cleaning station. There are only one or two 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.
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<gallery widths=216px>
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File:SPW801.jpg|1 x SPW801 adjustable spanner wrench
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</gallery>
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<center>
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[[Image: 140729_OpticsBootcamp_05.jpg|frameless|x200px]]
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[[Image: 140729_OpticsBootcamp_07.jpg|frameless|x200px]]
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</center>
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* Mount the LED between two SM2RR retaining rings in an LCP01 cage plate.
 +
** Screw in one SM2RR to a depth of 1 mm.
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** Run the wires of the LED through the opening in the LCP01 and insert the LED until it is resting on the retaining ring. It will get sandwiched in-between two retaining rings.
 +
** Add a second SM2RR retaining ring to secure the LED. Use the SPW801 adjustable spanner wrench or a small flat bladed screwdriver to tighten the retaining ring.  The SPW801 can be opened until its width matches the SM2RR diameter, the separation between the ring's notches.
 +
 
 +
<center>
 +
[[Image: LensInLensTube.JPG|frameless|x200px]]
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</center>
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* Place the 35 mm lens in a SM1L05 lens tube.
 +
** Don't just drop the lens in. Use lens paper to gently lower the lens into the tube.
 +
** The lens will rest against a ridge inside of the tube, near the threaded end.
 +
** Don't touch the lens while you are putting it in.
 +
* Start threading an SM1RR retaining ring into the tube and then tighten it with the red SPW602 spanner wrench.
 +
* Thread an SM1RR retaining ring in another SM1L05 lens tube and use the SPW602 spanner wrench to drive it about 90% of the way down the tube.
 +
* Place the 25 mm lens in a SM1L05 lens tube with its curved side facing the external threads of the lens tube.
 +
* Thread a second SM1RR retaining ring into the lens tube and tighten it with the SPW602 spanner wrench.
 +
 
 +
<center>
 +
[[Image: LensTubeLCP02.JPG|frameless|x200px]]
 +
</center>
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* Screw the lens tube with the 25 mm lens a into another LCP02. Screw the ND filter into the other side of the LCP02.
 +
* Connect the LCP02 and the LCP02 together using cage rods
 +
 
 +
<center>
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[[Image: 140730_OpticsBootcamp_1.jpg|frameless|x200px]]
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[[Image: 140730_OpticsBootcamp_2.jpg|frameless|x200px]]
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</center>
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*Connect the LED
 +
** Turn the power supply on.
 +
** Make sure the power supply is not enabled (green LED below the OUTPUT button is not lit).
 +
** Use the righthand set of knobs to set the current and voltage
 +
*** Adjust the CH1/MASTER VOLTAGE knob so the display reads about 5 Volts.
 +
*** Adjust the CH1/MASTER CURRENT knob to so the display reads 0.1 Amps.
 +
*** IMPORTANT: Never set the CURRENT to a value greater than 0.5A, as this will burn out the LED.
 +
** Connect the + (red) terminal of channel CH1 on the power supply to the red wire of the LED.
 +
** Connect the - (black) terminal of channel CH1 on the power supply to the black wire of the LED.
 +
** Press the OUTPUT button to enable the power supply and light the LED.
 +
** Adjust the LED brightness using the power supply's CURRENT knob.
 +
*Collimate the illuminator
 +
**Slide the lens along the cage rods until the light from the LED focuses at some point far away (like on the wall)
 +
**Tighten the set screws to keep the lens a fixed distance from the LED. There is no need to overnighted the screws, just make sure that the lens doesn't slide around or move relative to the LED.
 +
 
 +
====Assemble the objective cage components ====
 
*** 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.
 
*** 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.
*** 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.)
+
***  
[[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]]
+
 
 
** 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!
 
** 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!
 
[[Image:130816_CageCube.png|center|thumb|400px|The mounting struts should remain recessed within the cage cube walls.]]
 
[[Image:130816_CageCube.png|center|thumb|400px|The mounting struts should remain recessed within the cage cube walls.]]
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** Each group will receive their own LED. Please ask an instructor if you cannot find one.
 
** Each group will receive their own LED. Please ask an instructor if you cannot find one.
 
{{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.}}
 
{{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.}}
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 +
====Assemble the remaining beam path====
 +
====Putting it all together====
  
 
<div id="MATLABsaving"></div>
 
<div id="MATLABsaving"></div>
==Recording, displaying and saving images in MATLAB==
+
====Recording, displaying and saving images in MATLAB====
 
<figure id="fig:UsefulImageAcquisition">
 
<figure id="fig:UsefulImageAcquisition">
 
[[Image:UsefulImageAcquisiton.png|thumb|right|<caption>The UsefulImageAquisition window can be used to (hopefully) easily control the camera settings. To run it, type "<tt>foo = UsefulImageAcquisition;
 
[[Image:UsefulImageAcquisiton.png|thumb|right|<caption>The UsefulImageAquisition window can be used to (hopefully) easily control the camera settings. To run it, type "<tt>foo = UsefulImageAcquisition;
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</ol>
 
</ol>
  
====Magnification measurement====
+
===Exercise 2: Measure the microscope's magnification ===
 
<figure id="fig:Manta_camera_side_view">
 
<figure id="fig:Manta_camera_side_view">
 
[[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]]
 
[[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]]
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# Using your magnification measurements, calculate the FOV of the microscope for each objective.
 
# Using your magnification measurements, calculate the FOV of the microscope for each objective.
  
====Particle size measurement====
+
===Exercise 3: Particle size measurement===
 
[[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!]]
 
[[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!]]
  
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# Measure and report the average size and uncertainty of the spheres in each sample. How many spheres should you measure?
 
# Measure and report the average size and uncertainty of the spheres in each sample. How many spheres should you measure?
  
====Microscope storage====
+
==Microscope storage==
  
 
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:
 
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:

Revision as of 18:14, 11 August 2017

20.309: Biological Instrumentation and Measurement

ImageBar 774.jpg


Mens et Manus.jpg

Assignment Details

Before the lab

  1. Snell's law:
    A laser beam shines on to a rectangular piece of glass of thickness $ T $ at an angle $ \theta $ of 45° from the surface normal, as shown in the diagram below. The index of refraction of the glass, ng, is 1.41 ≈√2. The index of refraction for air is 1.00.
    Optics bootcamp snells law problem.png

    (a) At what angle does the beam emerge from the back of the glass?

    (b) When the beam emerges, in what direction (up or down) is it displaced?
    Optional:
    (c) By how much will the beam be displaced from its original axis of propagation?

  2. Chelonian size estimation:
    In the diagram below, an observer at height $ S $ above the surface of the water looks straight down at a turtle swimming in a pool. The turtle has length $ L $, height $ H $, and swims at depth $ D $.
    Turtle problem.png

    (a) Use ray tracing and Snell's law to locate the image of the turtle. Show your work.
    (b) Is the image real or virtual?
    (c) Is the image of the turtle deeper, shallower, or the same depth as its true depth, $ D $?
    (d) Is the image of the turtle longer, shorter, or the same length as its true length, $ L $?
    (e) Is the image of the turtle taller, squatter, or the same height as its true height, $ H $?

  3. Ray tracing with thin, ideal lenses:
    Lenses L1 and L2 have focal lengths of f1 = 1 cm and f2 = 2 cm. The distance between the two lenses is 7 cm. Assume that the lenses are thin. The diagram is drawn to scale. (The gridlines are spaced at 0.5 cm.) Note: Feel free to print out this diagram so you can trace the rays directly onto it. Or maybe use one of those fancy tablet thingies that the kids seem to like so much these days.

    RayTracing1.png


    (a) Use ray tracing to determine the location of the image. Indicate the location on the diagram.
    (b) Is the image upright or inverted? Is the image real or virtual?
    (c) What is the magnification of this system?

    Optional:

    (d) Lens L1 is made of BK7 glass with a refractive index n1 of 1.5. Lens L2 is made of fluorite glass with a refractive index n2 of 1.4. Compute the focal lengths of L1 and L2 if they are submerged in microscope oil (refractive index no = 1.5).

  4. Measuring focal lengths (hint: this will be really useful later):
    In the two-lens system shown in the figure below, the rectangle on the left represents an unspecified lens L1 of focal length $ f_1 $ separated by 0.5 cm from another lens L2 with focal length $ f_2 $ of 1 cm.
    UnknownLens.png

    Find the value of $ f_1 $ such that all the rays incident parallel on this system will be focused at the observation plane, located at a distance d of 2 cm away from L2.

In the lab

Work through the lab instructions below and answer the following questions along the way.

Part 1: getting oriented

  1. Report the focal lengths of the mystery lenses A through D.
  2. For 5 different object distances, measure the image height, and image distance. Plot (1/si vs. 1/f - 1/s0, as well as hi/ho vs si/so. Do the relationships between M, Si and So match the theory?
  3. Measure the noise spectrum of the MantaG CCD camera. Plot pixel variance vs. pixel mean on a log-log scale. Did the plot look the way you expected? How does noise vary as a function of light intensity?

Part 2: building the microscope

  1. Assemble your microscope
  2. Record an image of the micro ruler at 10x and 40x magnification. Record an image of the ronchi ruler at 100x magnification. Save these images as .mat files so that you can analyze them after the lab.

After the lab

  1. In a table, report the object and image height, nominal and actual magnifications, and field of view for each objective
  2. Measure the size of the ‘3um beads’. Include a recorded image of the beads. Report the measured bead size and appropriate error.


Lab Instructions, Part 1: getting oriented

Lab Orientation

Welcome to the '309 Lab. Before you get started, take a little time to learn your way around.

20.309 Lab.jpg

Thorlabs CP02 vs CP02T

The 20.309 lab contains more than 15,000 optical, mechanical, and electronic components that you may choose from in order to complete your assignments throughout the semester. You can waste a lot of time looking for things, so take a little time to learn your way around. The floor plan below shows the layout of rooms 16-352 and 16-336. When you get to the lab, take a walk around, especially to the center cabinet, west cabinet, and west drawers. Go everywhere and check things out for a few minutes. Read the time machine poster near the printer. Discover the stunning Studley tool chest poster near the east parts cabinet.

You probably noticed that many components are filed in boxes labeled with a sequence of letters and numbers. A company called Thorlabs manufactures the most of the optics and optical mounts you will use. The lab manuals frequently refer to components by their part number, which is usually one or two letters that have a vague relationship to the name of the part, followed by a couple of numbers. For example, the Thorlabs part number for a certain type of cage plate is "CP02." Thorlabs makes many kinds of cage plates, each with a subtle variation in its design. All of the cage plate part numbers starts with the letters "CP," followed by some numbers and in some cases a letter or two at the end. The Thorlabs website has an overview page of various kinds of cage plates here. Googling "Thorlabs CP02" is a quick way to get to the catalog page for that part.

To the untrained eye, many of the components in the lab are indistinguishable. The picture on the right shows just one example of strikingly-similar-looking bits. Take care when you select components from stock. Students sometimes return components to the wrong bins. Use even more care when you put things away. It is helpful to sing the One of These Things is Not Like the Others song from Sesame Street out loud while you are putting things away to ensure that you don't make any errors. (For the final exam, you will be required to identify various components while blindfolded.)


Map of the 20.309 lab.

Note that Station 9 is now the "Instructor Station," and the previously labeled instructor station is now "Station 5" and available for your use.

See! Wasn't that a worthwhile journey?


Exercise 1: measure focal length of lenses

Make your way to the lens measuring station. Measure the focal length of the four lenses marked A, B, C, and D located nearby.

Exercise 2: imaging with a lens

Figure 1: Imaging apparatus with illuminator, object, lens, and CCD camera mounted with cage rods and optical posts.

Make your way to the pre-assembled lens setup that looks like the thing in Figure 1. You will be using similar parts to build your microscope, so take some time to look at the construction of the apparatus. Try to identify the illuminator, the object (a glass slide with a precision micro ruler pattern on it), the imaging lens, and the camera (where the image will be captured). All of the components are mounted in an optical cage made out of cage rods and cage plates. The cage plates are held up by optical posts inserted into post holders that are mounted on an optical table. You will be able to adjust the positions of the object and lens by sliding them along the cage rods.

In this part of the lab, you are going to use this simple imaging system with one lens to compare measurements of the object distance $ S_o $, image distance $ S_i $, and magnification $ M $ with the values predicted by the lens makers' formula:

$ {1 \over S_o} + {1 \over S_i} = {1 \over f} $
$ M = {h_i \over h_o} = {S_i \over S_o} $

Measure stuff

  • Start with the object at about 2$ f $ = 70 mm from the lens.
  • Launch MATLAB
  • In the command window, start the image acquisition tool by running the following two lines of code:
foo = UsefulImageAcquisition;
foo.Initialize
  • After the Image Acquisition window appears, click the Start Preview button.
  • Set the exposure time
    • In the lower right side of the window, edit Exposure to obtain a well-exposed image. The units are in microseconds.
    • You can keep the default values for the Gain, Frame Rate, and Number of Frames for now.
    • You may also need to adjust the current in your LED. Remember not to exceed 0.5 A.
  • Move the lens and the object to produce a focused image.
Figure 2: Side view of the Matna G-032 camera. The detector is recessed inside the body of the camera, 17.5 mm from the end of the housing. The detector comprises an array of 656x492 square pixels, 7.4 μm on a side. The active area of the detector is 4.85 mm (H) × 3.64 mm with a diagonal measurement of 6 mm. The manual for the camera is available online here.
  • When everything is set, measure the distance $ S_o $ from the target object to the lens and the distance $ S_i $ from the lens to the camera detector.
    • Figure 7 shows the location of the detector inside of the camera.
  • In the image acquisition window, press Acquire (lower right).
  • Save your image to the MATLAB workspace
    • Your image is saved in foo.ImageData but it will be overwritten next time you acquire an image. You will need to copy the image into a new variable before continuing.
    • Return to the MATLAB console window.
    • Choose a descriptive variable name such as Image1 and save your data to it:
Image1 = foo.ImageData;
  • Compute the magnification.
    • In the MATLAB Command Window, display your image by typing figure; imagesc( Image1 ); colormap gray;
    • Go back to the command window (by pressing ALT-TAB on the keyboard or selecting it from the task bar) and type imdistline.
    • Use the line to measure a feature of known size on the micro ruler
      • The small tick marks are 10 μm apart
      • The large tick marks are 100 μm apart
      • The whole pattern is 1 mm long.
      • The pixel size is 7.4 μm
  • Does your value agree with the ones predicted by the lens makers' formula?

Repeat for several image and object distances and plot the results

  • Repeat the procedure at several image and object distances. Do an equal number with the target placed at less than and greater than 2$ f $ from the lens.
  • Record your measurements
  • Plot the results
    • Plot $ {1 \over S_i} $ as a function of $ {1 \over f} - {1 \over S_o} $.
    • Plot $ {h_i \over h_o} $ as a function of $ {S_i \over S_o} $.
  • Do the relationships between $ M $, $ S_o $, and $ S_i $ match the theory?
  • What sources of error affect your measurements?

Exercise 3: noise in images

Almost all measurements of the physical world suffer from some kind of noise. Capturing an image involves measuring light intensity at numerous locations in space. The Manta G-032 CCD cameras in the lab measure about 320,000 unique locations for each image. Every one of those measurements is subject to noise. In this part of the lab, you will quantify the random noise in images made with the Manta cameras, which are the same ones that you will use in the microscopy lab.

Figure 3: Noise measurement experiment. The cameras in the lab produce images with 656 horizontal by 492 vertical picture elements, or pixels. At regular intervals, the camera measures the intensity of light falling on each pixel and returns an array of pixel values $ P_{x,y}[t] $. The pixel values are in units of analog-digital units (ADU).

So what is noise in an image? Imagine that you pointed a camera at a perfectly static scene — nothing at all is changing. Then you made a movie of, say, 100 frames without moving the camera or anything in the scene or changing the lighting at all. In this ideal scenario, you might expect that every frame of the movie would be exactly the same as all the others. Figure 3 depicts a dataset generated by this thought experiment as a mathematical function $ P_{x,y}[t] $. If there is no noise at all, the numerical value of each pixel in all 100 of the images would be the same in every frame:

$ P_{x,y}[t]=P_{x,y}[0] $,

where $ P_{x,y}[t] $ is the the pixel value reported by the camera of at pixel $ x,y $ in the frame that was captured at time $ t $. The square braces indicated that $ P_{x,y} $ is a discrete-time function. It is only defined at certain values of time $ t=n\tau $, where $ n $ is the frame number and $ \tau $ is the interval between frame captures. $ \tau $ is equal to the inverse of the frame rate, which is the frequency at which the images were captured.

You probably can guess that IRL, the frames will not be perfectly identical. We will talk in class about why this is so. For now, let's just measure the phenomenon and see what we get. A good way to make the measurement is to go ahead and actually do our thought experiment: make a 100-frame movie of a static scene and then see how much each pixel varies over the course of the movie. Any variation in a particular pixel's value over time must be caused by random noise of one kind or another. Simple enough.

(An alternate way to do this experiment would be to simultaneously capture the same image in 100 identical, parallel universes. This will obviously reduce the time needed to acquire the data. You are welcome to use this alternative approach in the lab.)

Figure 4: Pixel variance versus mean mystery plot. Can you stand the suspense?

We need a quantitative measure of noise. Variance is a good, simple metric that specifies exactly how unsteady a quantity is, so let's use that. In case it's been a while, variance is defined as $ \operatorname{Var}(P)=\langle(P-\bar{P})^2\rangle $.

So here's the plan:

  • Point your camera at a static scene that has a range of light intensities from bright to dark.
  • Make a movie.
  • Compute the variance of each pixel over time.
  • Make a scatter plot of each pixel's variance on the vertical axis versus its mean value on the horizontal axis, as shown in Figure 4.

Plotting the data this way will reveal whether or not the quantity of noise depends on intensity. With zero noise, the plot would be a horizontal line on the axis. But you know that's not going to happen. How do you think the plot will look?

Set up the scene and adjust the exposure time

The first step is to set up a scene with a large range of intensities. It's also important to measure roughly the same number of pixels for each intensity value. How can we diagnose the intensity range and number of pixels at each intensity? Enter the histogram. This useful plot will tell you the distribution of pixel intensities in your image. In our case, we will use a histogram to help us maximize the range and even out the number of pixels at each intensity.

For this part of the lab, you will change your experimental set up to obtain an image with an approximately uniform intensity histogram. Lucky for you, the UsefulImageAcquisition tool displays the intensity histogram of your image in the top right corner of the Image Acquisition window. It is up to you to decide how to get the best image, but here are a few guidelines:

Figure 5: Example intensity histogram with approximately uniform distribution of pixel values over the range 10-2000 ADU.
  • In the Image Acquisition window, click Start Preview.
  • Set the Exposure property to 100.
  • Adjust your experiment until your histogram is a roughly uniform distribution of pixel values between about 10 and 2000. Figure 5 shows a reasonably nice histogram. You might want to try some combination of sliding the optical components around, and adjusting the exposure time and LED current (remember not to exceed 0.5A!).
  • Make a schematic including relevant distances of your final setup.

If you ever want to plot your own histogram from a saved image, here is some example MATLAB code to do just that:

[ counts, bins ] = hist( double( squeeze( exposureTest(:) ) ), 100);
semilogy( bins, counts, 'LineWidth', 3 )
xlabel( 'Intensity (ADU)' )
ylabel( 'Counts' )
title( 'Image Intensity Histogram' )

If you're curious, the hist MATLAB function takes in the intensities you measured in the image exposureTest, divides the maximum range of intensities into a given number of equally spaced intervals or 'bins' (100 in this case), then counts the number of pixels which fall into each bin.

Acquire movie and plot results

Once you are set up correctly, make a movie and plot the results using the procedure below:

  1. Capture a 100 frame movie.
    1. Go to the Image Acquisition window and change the Number of Frames property from 1 to 100.
    2. Press Acquire.
    3. Switch to the MATLAB console window. (Press alt-tab until the console appears.)
    4. Save the movie to a variable called noiseMovie by typing: noiseMovie = foo.ImageData;
  2. Plot pixel variance versus mean.
    1. Use the code below to make your plot.
pixelMean = mean( double( squeeze( noiseMovie) ), 3 );
pixelVariance = var( double( squeeze( noiseMovie) ), 0, 3 );
[counts, binValues, binIndexes ] = histcounts( pixelMean(:), 250 );
binnedVariances = accumarray( binIndexes(:), pixelVariance(:), [], @mean );
binnedMeans = accumarray( binIndexes(:), pixelMean(:), [], @mean );
figure
loglog( pixelMean(:), pixelVariance(:), 'x' );
hold on
loglog( binnedMeans, binnedVariances, 'LineWidth', 3 )
xlabel( 'Mean (ADU)' )
ylabel( 'Variance (ADU^2)')
title( 'Image Noise Versus Intensity' )

(Need a refresher on loglog plots? Click here.)

Lab Instructions, Part 2: building a microscope

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.

—Animadversions on the Machina Coelestis of Johannes Hevelius, 1674

Don't you just buy a [expletive deleted] microscope?

Anonymous 20.309 student, Fall 2007

Background materials and references

In this part of the lab you will jump right in to building a full-fledged microscope. The following online materials provide useful background.

Exercise 1: Assemble the microscope

Microscope block diagram

To get you started, here is a block diagram of a 20.309 microscope. Note that for Assignment 1, you will be building the brightfieqld (or transillumination) path. You should leave space for the fluorescence path, which you will complete next week.

20.309 microscope block diagram

Optical components

Below is a brief introduction to a few of the different components comprising your microscope. Various systems for optical construction are 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 ThorLabs. The structure should be rigid, and the components sufficiently tightened so that your optics remain aligned even after moving your microscope. Understanding how all of the components work together can be daunting. Ask about any components that perplex you.

Lenses

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.

  • Tip: Verify all optics before you use them by determining the focal length with a ruler. You can use the lens measuring station. Alternatively, you can use the ceiling fluorescent lamps as a light source and measure 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)?
  • 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.
  • Handle lenses only by the edges. If a lens is dirty, first remove grit with a blast of clean air or CO2. 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.
Objective lenses

Please see the Nikon Introduction to Microscope Objectives at their excellent MicroscopyU website.

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.

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.
  • 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.
  • The 100× objective is designed to be used with immersion oil. When using the 100× objective, place a drop of oil directly on the tip on the objective. Bring the drop in contact with the slide cover glass. After use, clean off the remaining oil by wicking it away with lens paper or a Kim-wipe. Do not put samples away dirty.
  • 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.
Sample stage

A precision Newport X/Y/Z stage[1] 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).

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.

CCD camera

The microscope you will build does not have an eyepiece for direct visual observation. Instead, images will be captured with a CCD camera[2]. 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.

Design

Sketch out the design for your microscope on paper (or print out the above diagram). For this part of the lab, you can just draw the bright field illumination path. Label all the optical elements (lenses, mirrors, microscope objectives, and camera), distances, lens specifications, and orientations.

  • 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×)?
  • Which sections of the light path can be open (strut-based structure, cage rods)? Which would better enclosed (Thorlabs lens tubes)?
  • In what way will the illumination LED color affect your design? your results?
  • Which lens will you use between the LED and the sample for bright field transmitted light imaging?

Next, take a look at the example microscope in the lab. This microscope is here for your reference, but please do not touch, alter, or remove parts from the example microscope.

The general layout of the 20.309 microscope is compact and stand-alone; it fits and can be transported onto a breadboard

Can you identify all the components of your diagram? Try to think about the purpose of each component, and why it is laid out a particular way. Once you're satisfied that you understand the big picture, it's time to build your own!

A few tips to keep in mind:

  • Reproduce the general layout of the example microscope: it grants compactness and allows your device to be a stand-alone breadboard-transportable microscope.
  • 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:

Assemble the base

Gather the following parts:

  • On a 1' x 2' x 1/2" optical breadboard, 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 above picture is in error as the P14 is only 9 positions from a short side of the breadboard.)

Assemble the illuminator

Gather parts:

Most of the tools you will need are located in the drawers next to your lab station. 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. Here is a list of the tools you will need:

You will also need to use an adjustable spanner wrench. The adjustable spanner resides at the lens cleaning station. There are only one or two 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.

140729 OpticsBootcamp 05.jpg 140729 OpticsBootcamp 07.jpg

  • Mount the LED between two SM2RR retaining rings in an LCP01 cage plate.
    • Screw in one SM2RR to a depth of 1 mm.
    • Run the wires of the LED through the opening in the LCP01 and insert the LED until it is resting on the retaining ring. It will get sandwiched in-between two retaining rings.
    • Add a second SM2RR retaining ring to secure the LED. Use the SPW801 adjustable spanner wrench or a small flat bladed screwdriver to tighten the retaining ring. The SPW801 can be opened until its width matches the SM2RR diameter, the separation between the ring's notches.

LensInLensTube.JPG

  • Place the 35 mm lens in a SM1L05 lens tube.
    • Don't just drop the lens in. Use lens paper to gently lower the lens into the tube.
    • The lens will rest against a ridge inside of the tube, near the threaded end.
    • Don't touch the lens while you are putting it in.
  • Start threading an SM1RR retaining ring into the tube and then tighten it with the red SPW602 spanner wrench.
  • Thread an SM1RR retaining ring in another SM1L05 lens tube and use the SPW602 spanner wrench to drive it about 90% of the way down the tube.
  • Place the 25 mm lens in a SM1L05 lens tube with its curved side facing the external threads of the lens tube.
  • Thread a second SM1RR retaining ring into the lens tube and tighten it with the SPW602 spanner wrench.

LensTubeLCP02.JPG

  • Screw the lens tube with the 25 mm lens a into another LCP02. Screw the ND filter into the other side of the LCP02.
  • Connect the LCP02 and the LCP02 together using cage rods

140730 OpticsBootcamp 1.jpg 140730 OpticsBootcamp 2.jpg

  • Connect the LED
    • Turn the power supply on.
    • Make sure the power supply is not enabled (green LED below the OUTPUT button is not lit).
    • Use the righthand set of knobs to set the current and voltage
      • Adjust the CH1/MASTER VOLTAGE knob so the display reads about 5 Volts.
      • Adjust the CH1/MASTER CURRENT knob to so the display reads 0.1 Amps.
      • IMPORTANT: Never set the CURRENT to a value greater than 0.5A, as this will burn out the LED.
    • Connect the + (red) terminal of channel CH1 on the power supply to the red wire of the LED.
    • Connect the - (black) terminal of channel CH1 on the power supply to the black wire of the LED.
    • Press the OUTPUT button to enable the power supply and light the LED.
    • Adjust the LED brightness using the power supply's CURRENT knob.
  • Collimate the illuminator
    • Slide the lens along the cage rods until the light from the LED focuses at some point far away (like on the wall)
    • Tighten the set screws to keep the lens a fixed distance from the LED. There is no need to overnighted the screws, just make sure that the lens doesn't slide around or move relative to the LED.

Assemble the objective cage components

      • 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.
    • 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!
The mounting struts should remain recessed within the cage cube walls.
  • 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.)
  • Check all your lenses for cleanliness before you use them. You'll save yourself some troubleshooting time and effort down the road!
  • Make sure all your components are "leveled" (horizontal, not slanted).
  • Use tube rings (and never an SM1T2, SM1V01, or SM1V05) to mount optics in lens tubes.
  • 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 — 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.
Adjustable Thorlabs SM1V05 and SM1T2 connectors precede the quick-connect union to the CCD camera.
  • 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).
Insertion and removal of optical components is facilitated by a three-strut-only link.
  • The Nikon objective lenses are designed to be paired with a 200 mm tube lens.
  • Assume that the objectives behave as ideal plano-convex lenses.
  • Fine focusing will be achieved by adjusting the height of the sample stage.
  • Tip: Throughout the optical microscopy lab, start the alignment with a 10× objective and then progress to 40× and 100×.
  • 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 and 3, and this can be quite useful when trying to bring a fluorescent sample into focus.
    • Each group will receive their own LED. Please ask an instructor if you cannot find one.


Warning.jpg 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.


Assemble the remaining beam path

Putting it all together

Recording, displaying and saving images in MATLAB

Figure 6: The UsefulImageAquisition window can be used to (hopefully) easily control the camera settings. To run it, type "foo = UsefulImageAcquisition; foo.Initialize" into the MATLAB console window.
  1. Run the UsefulAcquisition tool
    • Launch MATLAB and in the command window type:
    • foo = UsefulImageAcquisition; 
      foo.Initialize 
    • The Image Acquisition window (Figure 6) will open with the controls for the camera.
    • The "Manta_G-032B" camera is configured 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.
    • In the Image Acquisition window set "Frame Rate" to 20. This will cause the camera to take 20 complete images per second (this will only be relevant when recording movies in Part 3 of the lab).
    • Click the "Start Preview" button. The live image from the camera should appear in the Preview pane.
    • If this does not produce a live image, use an appropriate expletive, and ask an instructor to figure out what the heck went wrong.
    • Change the "Exposure" setting to produce a good image. The value sets the exposure time for each frame in microseconds.
  2. Recording an image
    • In the Image Acquisition window, set "Number of Frames" to 1. This setting controls how many images MATLAB will record each time you press the "Acquire" button.
    • When you are happy with the image displayed by the live preview, press "Acquire". The live preview will stop.
    • The image is now stored in the foo.ImageData variable, which will update each time you acquire a new image. To copy the data into a new variable, choose a descriptive name for your image like 'microruler10x' and save it to your workspace by typing the following into the MATLAB command window:
    • microruler10x = foo.ImageData;
    • Next, in the MATLAB command window type
    • whos microruler10x 
    • This command will display relevant information about the new variable you’ve created. You should see that the image is represented as a 492x656 matrix of 16-bit integers.
  3. Displaying the image
    • You can display images using a variety of commands in MATLAB. In the optics bootcamp we used the imagesc command which scales the image intensity (the brightest pixel is white, the darkest is black). In some cases, like when you have very dim images, this command can be misleading. It’s better to use the un-scaled imshow command for quantitative measurements.
    • 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, while your 12-bit image only contains values from 0-4095. This leaves a considerable portion of the number range unoccupied. Consequently, if you type imshow( microruler10x ), you will see an image that looks almost completely black (try it!).
    • One way to make this work is to rescale your image to 16 bits: imshow( 65535/4095 * microruler10x )
    • An even better way to work with images in MATLAB is to convert them to double precision floating point format straightaway. Double precision floating point numbers can represent an extremely wide range of values with high precision. Convert the image to a double and rescale it using the following command:
    • microruler10x = double( microruler10x ) / 4095; 
    • This conversion has made your image into a double with a range of intensities from 0-1, with 1 being full intensity and 0 completely dark. Now try:
    • whos microruler10x
      imshow( microruler10x ) 
  4. Saving your image
    • Save images in a .mat format so that you can easily reload them into Matlab for later use.
    • save microrulerImages     % saves entire workspace to filename 'microrulerImages.mat'
      save microrulerImages image1 image2     %saves only variables image1 and image2 to filename 'microrulerImages.mat'
    • To reload your data the next time you open matlab, navigate to the folder where you saved your workspace, type
    • load microrulerImages 
    • If you want to save individual images as a .PNG (a good format for use in your report or other programs), the command might look something like:
    • imwrite( im2uint16( microruler10x ), 'microruler10x.png', 'png' );
    • Note that you can also use the File→Save As menu after displaying an image or figure. This is useful if you want to save additions to your images (like data cursors). However, we recommend always saving the raw data as .mat files so that you are able to re-process your images later.

Exercise 2: Measure the microscope's magnification

Example images included by past students in their Week 1 report: (top) Air Force target, (center) Silica spheres and dust, (bottom) Ronchi Ruling

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.

  1. Start the live preview using the UsefulImageAcquisition tool
  2. Ensure that the camera's field of view is approximately centered in the objective's field of view.
    • 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.
  3. Start with the 10x objective and a microruler calibration slide.
    • The microruler calibration slide has tick marks that are 10 um apart. Every 100 um, there is a longer tick mark.
    • Make sure that the side of the microruler with the pattern on it faces the objective. Imaging through the thick glass causes distortion and many other troubles.
  4. Record an image of the microruler.
  5. Use imdistline or the data cursor to measure a known distance between rulings in your image and compute the magnification.
    • When choosing a distance to measure, consider the factors that influence the uncertainty of your measurement.
  6. Repeat the magnification measurement for the 40x and 100x objectives.
    • With the 100x objective, you may want to substitute the microruler with a Ronchi Ruling, a grating with 600 line pairs per millimeter. Why is it not wise to use the Ronchi Ruling with the 10x objective?
  7. Save your images in a .mat file for later use in MATLAB or as a PNG image for use in your report or other programs.
  8. Using your magnification measurements, calculate the FOV of the microscope for each objective.

Exercise 3: Particle size measurement

Example image of 3.2 μm beads using the instructor microscope. Submit picture to replace this!

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.

  1. Image 7.2 μm, 3.2 μm and 1 μm silica microspheres as described in the magnification measurement procedure (40x objective only).
  2. Measure and report the average size and uncertainty of the spheres in each sample. How many spheres should you measure?

Microscope storage

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:

  • 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.
  • Keep all of the boxes for the optics you use with your instrument to simplify putting things away.
  • Take a blue bin to store loose items (such as lens boxes) in.
  • Stages, CCD cameras, neutral density filters and barrier filters stay at the lab station. Do not store these with your microscope.
  • Return objective lenses to the drawer when you are not using them. (Do not store them with your microscope.)
  • The stages are very expensive. Always lift from the bottom.
  • 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.

References

  1. Precision Newport X/Y/Z stages
  2. Allied Manta G032B
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