Optical Microscopy Part 1: Brightfield Microscopy
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
Overview
In the first week of the microscopy lab, you will construct a brightfield microscope.
Background materials and references
The following online materials provide useful background for this part of the microscopy lab.
- Geometrical optics and ray tracing
- Physical optics and resolution
- Lectures 1 through 9 of the 20.309 class
- From Nikon MicroscopyU
- Conjugate planes in optical microscopy (includes transmitted and reflected (epi) illumination)
- Snell's law
- Resolution
Components of the 20.309 microscope
Optical construction
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. There are lots of constraints.
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 ThorLabs. Understanding how all of the components in the catalog work together is 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. 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)?
- 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 for a 200 mm tube lens. An adapter ring converts the objective mounting threads to the SM1 threads used by the lens tube system.
- 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.
- 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.
- 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.
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.
Microscope construction
Design
Sketch out a rough design for your microscope on paper. Begin with the bright field illumination path.
- 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)? Which would better enclosed (Thorlabs 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?
Practice
- Please do not remove parts from the example microscope.
- On a 1' x 2' x 1/2" 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.
- 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.
- 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!
- Set the distance between the top of the breadboard and the top of the upper CP02 to 12.7 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.
- 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.
- 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.
- 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 but progress to 40× and 100×.
- You can use either a red or a blue LED illuminator for bright-field transmitted light imaging.
- Each group will receive their own LED. Please ask an instructor if you cannot find one.
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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. |
Magnification measurement
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.
- Use MATLAB's Image Acquisition Tool to view a live display and record images
- Launch Matlab and type imaqtool. An Image Acquisition Tool window should fill the screen.
- Select the "Mono12" mode of the "Manta_G-032B (gentl-1)" camera in the Hardware Browser pane.
- 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.
- 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.
- 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, 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.
- 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.
- 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.
- Measure the magnification of your microscope using the 10x, 40x, and 100x objectives.
- Start with the 10x objective and an Air Force imaging target.
- 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 [this Wikipedia page].
- 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.
- 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).
- Why is it not judicious to image the Ronchi ruling with the 10x objective?
- 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.
- Press the "Start Acquisition" in the Preview Pane. (The live preview will stop.)
- 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 AirForce14lp10x. Data from the image you acquired will be available in the Matlab workspace.
- Switch to the MATLAB command window and type whos AirForce14lp10x. The image is represented as a 492x656 matrix of 16-bit integers.
- To display the image, use the imshow command.
- 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 imshow( AirForce14lp10x ), you will see an image that looks almost completely black. Adjust the image to fill the full range by typing imshow( 16.0039 * AirForce14lp10x ). 16.0039 equals 16 + 1/256. This factor maps values in the range 0-4095 to 0-65535.
- 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. Matlab includes a function for converting images, im2double. Type AirForce14lp10x = im2double( 16.0039 * AirForce14lp10x ); to make the conversion and then use imshow 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.
- Use the data cursor to measure magnification
- When choosing the size of lines to image, consider the factors that influence the uncertainty of your measurement.
- Save your image as a .mat for later use in MATLAB (save AirForce14lp10x) 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: imwrite( im2uint16( AirForce14lp10x ), 'AirForce14lp10x.png', 'png' );.
- Start with the 10x objective and an Air Force imaging target.
- Repeat the magnification measurement for the 40x and 100x objectives.
- 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.
- Calculate the FOV of the microscope using all three objectives.
Particle size measurement
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.
- Image 7.2 μm, 3.2 μm and 1 μm silica microspheres as described in the magnification measurement procedure (40x objective only).
- 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.
Report outline
Find and follow all guidelines on the Microscopy report outline wiki page.
- Apparatus:
- Include a block diagram of your microscope, including all optical elements and relevant distances. It is not necessary to document the details of the mechanical construction.
- Describe your design calculations and considerations.
- Why not put in a nice snapshot of your ‘scope? (optional, but certainly a cherished memory in the making)
- Magnification
- Procedure
- Document the samples you used and how you captured images (camera settings, software used, etc…)
- Data
- Include example images.
- Analysis and Results
- Report the nominal and actual magnifications and fields of view you measured for the three objectives in a table. Report the length and width of the FOV (in distance units), not its area (in distance units squared).
- Document the method you used to find magnification.
- List the error sources that contributed significantly to systematic error and uncertainty in your measurement. To the degree possible, quantify the type and magnitude of the error.
- Discussion (optional for magnification measurement)
- Explain any challenges you faced in the magnification measurement.
- Procedure
- Particle diameter measurement
- Procedure
- Document the samples you used and how you captured images (camera settings, software used, etc…)
- Data
- Include example images.
- Analysis and Results
- Report the average size of the microspheres in each sample and a measure of variation.
- Describe how you measured the microspheres.
- List the error sources that contributed significantly to systematic error and uncertainty in your measurement. To the degree possible, quantify the type and magnitude of the error.
- Discussion
- Explain any challenges you faced measuring the size of silica microspheres.
- How did your measurements differ from the manufacturer's specified values? What factors contributed to the difference?
- Procedure
Optical microscopy lab
Code examples and simulations
- Converting Gaussian fit to Rayleigh resolution
- MATLAB: Estimating resolution from a PSF slide image
- Matlab: Scalebars
- Calculating MSD and Diffusion Coefficients
Background reading
- Geometrical optics and ray tracing
- Physical optics and resolution
- Optical aberrations
- Aperture and field stops
- Optical detectors, noise, and the limit of detection
- Manta G032 camera measurements
- Understanding log plots