Counting dots within nuclei

cellprofiler

#1

I am doing some FISH analysis to assess ploidy in cells by counting the number of copies of a given chromosome. I’d be interested in using Cell Profiler to do the counting for me. Basically, I would need to count the number of blue nuclei in a field, then count the number of green or red dots (each representing one chromosome) within each nucleus to get a
quantitation of the number of nuclei within the field that have one, two, three, four, etc., copies of that chromosome. Do you know if Cell Profiler could do this, and if there are any modules out there already? I saw one on the web page for counting nuclear speckles, but it seems like what it does is give the number of nuclei and the total number of speckles in the field, which is not exactly what I’m looking for.

Andy


#2

Hello Andy,
Indeed, it will work for counting individual speckles in each nucleus, not just each each speckle in an entire image. After running the pipeline, you can export the data into an excel file by clicking on Data Tools (in the main CellProfiler window) >> Export Data. It will prompt you to select the output file. Then, it will ask you which sets of data to export: export the “nuclei” and “image” measurements. The nucleus file contains one row of data for each nucleus, and one of the measured feature columns is “number of h2ax” (which is what we call the speckles; you can rename it, of course). Image file contains one row per image and one of the columns is “number of h2ax” which is the AVERAGE number of h2ax speckles per nucleus for all nuclei in that image. If you would like to view the measurements for EACH “h2ax” speckle (size, intensity, etc), all you need to do is select “h2ax” measurement when exporting the data; it contains one row of data for each speckle.

As you can see, using the example pipeline as is will give you the number of green speckles per nucleus, and so the major adjustment you will make is to similarly identify red speckles and Relate them. Please let me know if you have any further questions or concerns. It is a nice example for you to start with before modifying it to suit your experiments.

Thank you for posting you question!

Martha