Assignment 1, Part 4: Measuring magnification and bead size

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20.309: Biological Instrumentation and Measurement

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I took a good clear piece of Cork, and with a Pen-knife sharpen'd as keen as a Razor, I cut a piece of it off, and thereby left the surface of it exceeding smooth, then examining it very diligently with a Microscope, me thought I could perceive it to appear a little porous; but I could not so plainly distinguish them, as to be sure that they were pores, much less what Figure they were of: But judging from the lightness and yielding quality of the Cork, that certainly the texture could not be so curious, but that possibly, if I could use some further diligence, I might find it to be discernable with a Microscope, I with the same sharp Penknife, cut off from the former smooth surface an exceeding thin piece of it, and placing it on a black object Plate, because it was it self a white body, and casting the light on it with a deep plano-convex Glass, I could exceeding plainly perceive it to be all perforated and porous, much like a Honey-comb, but that the pores of it were not regular; yet it was not unlike a Honey-comb in these particulars.

I told several lines of these pores, and found that there were usually about threescore of these small Cells placed end-ways in the eighteenth part of an Inch in length, whence I concluded there must be neer eleven hundred of them, or somewhat more then a thousand in the length of an Inch, and therefore in a square Inch above a Million, or 1166400. and in a Cubick Inch, above twelve hundred Millions, or 1259712000. a thing almost incredible, did not our Microscope assure us of it by ocular demonstration.

Robert Hooke from Micrographia: or Some Physiological Descriptions of Minute Bodies made by Magnifying Glasses with Observations and Inquiries Thereupon (1665)[1]



Assemble the 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:


Gathering materials

You can spend a huge amount of time walking around the lab just getting things in the lab, so it makes sense to grab as many parts as possible in one trip.

The materials you will need for this lab are shown in the image gallery below, including a part number and descriptive name of each component. When you come across a part name or number that is not-all-that-self-explanatory, remember that google is your friend. Most of the parts are manufactured by a company called ThorLabs. If you ever have a question about any of the components, the 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 a handsome photo and detailed specifications.

Figure 1: Screw measuring gauge and example screw specifications.

Screw sizes are specified as <diameter>-<thread pitch> x <length> <type>. The diameter specification is confusing. Diameters ¼" and larger are measured in fractional inches, whereas diameters smaller than ¼" are expressed as an integer number that is defined in the Unified Thread Standard. The thread pitch is measured in threads per inch, and the length of the screw is also measured in fractional inch units. So an example screw specification is: ¼-20 x 3/4. Watch this video to see how to use a screw gauge to measure screws. (There is a white, plastic screw gauge located near the screw bins.) The type tells you what kind of head the screw has on it. We mostly use stainless steel socket head cap screws (SHCS) and set screws. If you are unfamiliar with screw types, take a look at the main screw page on the McMaster-Carr website. Notice the useful about ... links on the left side of the page. Click these links for more information about screw sizes and attributes. This link will take you to an awesome chart of SHCS sizes.

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.

Assemble the base

Gather the following parts:

  • Attach the PB1 base to the P14 post using a 1/4-20 screw.
  • 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.)
  • Secure the post base into the breadboard with 1/4-20 screws.

MicroscopeBase.jpg

Assemble the illuminator

Gather parts:

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.

  • 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.

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  • If you're using a lens with f=25mm, thread an SM1RR retaining ring into a SM1L05 lens tube and use the SPW602 spanner wrench to drive it about 90% of the way down the tube. If you're using a lens with a larger focal length, this step is unnecessary and you can place the lens directly at the back of the tube.
  • Place the condenser lens in the SM1L05 lens tube with its curved side facing the external threads of the tube.
    • Don't just drop the lens in. Use lens paper to gently lower the lens into the tube.
    • Don't touch the lens while you are putting it in.
  • Thread a second SM1RR retaining ring into the lens tube and tighten it with the SPW602 spanner wrench.
  • Screw the lens tube into an LCP02

LensInLensTube.JPG LensTubeLCP02.JPG

  • Connect the LCP01 and the LCP02 together using cage rods.
    • Use the tiny 4-40 set screws to secure the ER2 cage rods to the LCP01. There is no need to overnighted the screws, just make sure that they are tight enough that the rods don't slide around.
    • Slide on the LCP02 containing the lens. If you're using a plano-convex lens, make sure that the curved side is oriented the way you want it to be. No need to tighten the LCP02 set screws quite yet - you will adjust the position of the lens in the next step.

Illuminator.jpg


  • Connect the LED
    • 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, which you will be doing in later assignments. This can be quite useful when trying to bring a fluorescent sample into focus.
    • 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.
    • Pick up a red and a black cable from the cable rake in the corner of the lab. I like the ones with the flat horseshoe on one end and the alligator clip on the other.
    • 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.


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.


    • Press the OUTPUT button to enable the power supply and light the LED.
    • Adjust the LED brightness using the power supply's CURRENT knob.

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  • Collimate the illuminator
    • Shine the LED light at some far-away point (like on the wall). Slide the lens along the cage rods until the light comes into focus. Remember that collimated light is the same as forming an image 'at infinity'
    • Tighten the set screws of the LCP02 to fix the lens-LED distance.
    • Turn off the LED power supply and disconnect the alligator clips for now.

IlluminatorCollimation.jpg

  • Screw in the C1500 clamp into the LCP01 holding the LED. You will attach the illuminator to the 1.5" post on the microscope base later.

Assemble the objective cage components

Hopefully, you are becoming more comfortable finding things around the lab and assembling optomechanical components. We will stop providing quite so detailed assembly instructions, but don't hesitate to ask an instructor if you are confused.

  • Gather the following components:
    • 1 x CM1P01 Cage Cube-Mounted Turning Mirror
    • 4 x ER05 0.5" Cage rods
    • 4 x ER1 1" Cage rods
    • 4 x ER2 2" Cage rods
    • 2 x LCP01 Cage plates
    • 1 x LCP02 Cage plate adapter
    • 2 x C1500 1.5" post mounting clamps
  • Screw in the ER05 and ER1 cage rods to the two faces of the CM1P01 cage cube
  • Attach the LCP02 cage plate adaptor to the CM1P01 cube (using the smaller ER05 cage rods)

ObjectiveAssembly1.jpg ObjectiveAssembly2.jpg

  • Secure the ER2 cage rods to one of the LCP01 cage plates.
  • Stack the CM1P01 cube onto the LCP01 assembly and secure the other LCP01 plate to the top end.
    • Make sure that the 8-32 threaded mounting holes of the LCP01s are on the same side. Orient the turning mirror cube so that, if the 8-32 holes are closest to you, the ER1 cage rods stick out on the right side of the assembly.

ObjectiveAssembly3.jpg ObjectiveAssembly4.jpg

  • Screw in the C1500 clamps into the 8-32 threaded holes of the LCP01 cage plates.
  • Slide the assembly onto the 1.5" post on the microscope's base.
    • Tighten the clamps so that the distance between the top of the breadboard and the top surface of the upper LCP01 is 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.
  • Mount the illuminator onto the 1.5" post above the objective assembly.

ObjectiveAssembly5.jpg ObjectiveAssembly6.jpg

Assemble the remaining beam path

Continue assembling your microscope using the example microscope as a reference. Keep the following tips and suggestions in mind as you go:

  • The stability of your microscope will become more and more important as you increase the complexity of your measurements through the semester. You want every component to be fixed to the breadboard and well-supported. Use 0.5" posts (TR2) mounted in PH2 post holders and secure them to the breadboard with the BA1 base. (Please use a washer when securing the BA1 to the breadboard.)

AttachBA1.JPG PostAndPostHolder.jpg

  • Insert the C6W cage cube that will later hold the dichroic mirror required for fluorescence imaging. 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.
  • 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.
  • 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.
  • 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×.

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
foo = UsefulImageAcquisition;
foo.Initialize
  1. 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.
  2. 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.
  3. Record an image of the microruler.
  4. 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.
  5. 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?
  6. 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 ( Click here to get detailed instructions on saving in matlab).
  7. Using your magnification measurements, calculate the FOV of the microscope for each objective.


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a table including the object and image height, nominal and actual magnifications, and field of view for each objective (10x, 40x, 100x)



Measure particle size

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.
  1. Hooke, R. Micrographia: or Some Physiological Descriptions of Minute Bodies made by Magnifying Glasses with Observations and Inquiries Thereupon London:Jo. Martyn, and Ja. Allestry, Printers to the Royal Society; 1665
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