Fluorescence Microscopy

Diana Libuda's lab studies DNA double-strand break repair in C. elegans worms. She uses microscopy to image the worms, using a specific fluoresence marker of double strand breaks. We are going to analyze some of her data and ask whether there is a difference in the number of double strand breaks between her control and experimental conditions.

For each condition, we have a collection of images that are slices through a worm. If you flip through them quickly, you pass through the worm. These images are stitched into short videos below.

Task

The red globs are chromosomes inside nuclei, the green dots are double strand breaks. Your goal is to count the number of double strand breaks per nucleus under these treatment conditions.

• Choose 5 nuclei from the control worm and 5 nuclei from the experimental worm.
• Count the number of bright green puncta in each nucleus.
• Report the mean and standard deviation of the number of puncta per chromosome between your 5 control and 5 experimental chromosomes.
• Are the differences you observed statistically significant? (A t-test seems like a reasonable way to go about this...)

Hints:

• Define a circular mask for each nucleus you want to study.
• The same puncta can occur across multiple slices, but not all puncta appear in all slices. (This is a 3D chromosome blob). You'll have to make sure you don't count the same point twice.

Data

The collection of images are available here:

https://www.dropbox.com/s/dry59l6h14cqkkj/worm-images.zip?dl=0

If you unzip the directory, it has the following files and directories.

• worm-images

  • experimental
    • z000.png
    • z001.png
    • ...
    • z066.png
  • control
    • z011.png
    • z012.png
    • ...
    • z054.png

• Each zxxx.png file is a slice.

• The R, G, and B channels have different information:
  • R: chromosome marker
  • G: double-strand break marker
  • B: nucleus marker