Cell Biology Sample Assignment

Following Problems:

  1. You are using a brightfield microscope.
    1. Which user adjustment(s) will lower contrast but increase resolution?
    2. Which user adjustment(s) will lower contrast and have little effect on resolution?
  2. You insert a biological sample onto a microscope stage and illuminate it monochromatically with 488 nm light. You have an imaging CCD array with a square pixel dimension of 6.7 μm that you insert into the normal orthoscopic plane for the retina. The maximum half-angle of light that can illuminate and be accepted from the specimen for this microscope is 45°.
    1. What is the minimum total magnification necessary for this microscope + imager to capture images at or near the fundamental resolution limit?
    2. Use words to justify the step-by-step logic of your numerical solution in problem 2a. If you made additional assumptions for your solution, describe them.
  3. You are given a planoconvex thin lens with an index of refraction of 1.5 and a radius of curvature of 1 cm.
    1. Design a simple microscope using the lens above to achieve the magnification. State clearly the magnification you are attempting to produce. If you decide that you cannot build a simple microscope to meet all of these design constraints, you may select an arbitrary value for one of the constrained parameters (i.e., magnification, index of refraction, radius of curvature) in order to complete a successful design.
    2. Sketch your design on an optical axis. Be sure to label all the critical parts, including all of the free parameters. You should provide enough information in your sketch so that you could give it to someone and they could build your microscope.
    3. Write the transfer matrix for this simple microscope. Give a symbolic representation and a numerical representation.
    4. What is the working distance of your microscope? Include a description of how you define working distance.
  4. Sitting at your brightfield microscope image acquisition rig with your sample on the stage, you capture an image that looks like the following on your computer screen:
    1. List every method you can think of to improve this image (it may help you to formalize your definition of 'improve'). If more than one method achieves the same result, select and describe the best one.
    2. Describe in words what your image (either the one given above or your improved image) would look like if you were to apply the following convolution kernel:

    2 1 0
    1 0 1
    0 1 2

  5. Two cytosolic proteins are believed to interact in a regulatory pathway. You would like to demonstrate that they interact. You design an experiment to test for such an interaction using fluorescence resonance energy transfer (FRET). List all the considerations you can think of that must be taken into account for this experiment in order to demonstrate convincingly that such an interaction takes place.
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