Assignment 3 Overview

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20.309: Biological Instrumentation and Measurement

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Assignment details

This assignment has 2 parts:

  1. Part 1: Use the epifluorescence microscope you built to image fixed biological samples, and use the flat-field correction code you wrote for Assignment 2 to address non-uniform illumination;
  2. Part 2: Explore interesting calculations and considerations to guide your experimental design with fluorescence.

Submit your work on Stellar in a single PDF file with the naming convention <Lastname><Firstname>Assignment3.pdf. Here is a checklist of all things you have to turn in:


Pencil.png Here is a comprehensive list of what you need to turn in:
  1. A figure with images of the stained cell samples with and without Cyto-D.
    • For each sample, create 1 figure with 5 panels.
    • The panels of the figure should be: A) unprocessed image; B) reference image; C) dark image; D) flat-field corrected image; and E) histogram.
    • In the caption, specify the exposure and gain settings. Each image should have a scale bar. State the dimension of the scale bar in the caption.
    • For panel E, plot histograms of the unprocessed, dark, reference, and corrected image on the same set of axes. Plot log10( count ) on the vertical axis and intensity on the horizontal axis. Use a line plot instead of a bar chart for the histogram.
  2. Image profile
    • For one reference, dark and cell image set, plot an intensity profile across the same diagonal. You may also use a bead image, along with it's unique reference and dark images. The intensity of your three images should be on the same scale, i.e., 0 to 65,535 or 0 to 1. Place all three profiles on a single set of axes for comparison. (Use the improfile command in MATLAB.)
  3. Discussion
    • How did your beam expander design affect your images?
    • What differences did you observe between the cells with and without CytoD?
  4. Answers to all questions in Assignment 3, Part 2.


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