Identification and outline of transcription sites

Hello,

I am a new user of cell profiler and I am creating outlines for nucleus and cytoplasm of neurons to be used in FISHquant. I figured that I was also able to identify the transcription sites using cell profiler.
Using primary objects, followed by measure image intensity and filter objects (maximal per object in the nucleus) I was able to identify and create an outline for one of the transcription site. But the problem is that it is giving me only THE brightest object in the nucleus and not the two brightest.

Can you please help me identify the function which can allow me to identify and create an outline for the two brightest objects in the nucleus?

Thank you

Celia

Hi @Celia,

Taking a look at the help for the FilterObjects module, the “Maximal Per Object” method will only identify the one object within parent object (your nuclei) with the maximum measurement value (in your case, one of the intensity measurements that you feel best captures the transcription site).

If you want to always get the two brightest spots within your nuclei, you could:

  • Filter the objects to get the brightest spot, like you’re already doing
  • Use those filtered objects as a mask over your original RNA objects to remove them from that image (can be done with the MaskObjects module - select Yes for invert the objects)
  • Repeat the MeasureObjectIntensity on your newly masked RNA objects that have every pixel except those that were part of the brightest transcription site
  • Then repeat the filtering process again on these masked objects to get the 2nd brightest transcription site per nucleus
  • You can then combine the results from these two FilterObjects modules using CombineObjects, if desired

I hope that helps! If it’s not, a sample image, pipeline, and screenshots to illustrate what you’re looking for / where the analysis fails always help us to help you.
Good luck!
Pearl

It’s working ! Thank you

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