Identifying dim objects with noisy background using Cy3

Hi CellProfiler team! First of all, thanks for creating this great software. It’s amazing.

The problem I keep running into is in regards to identifying cells with cytoplasmic myosin6 stained by Cy3. Our stains usually contain noisy background that have similar intensities to our target cells. I’ve been playing with different modules including the CorrectIllumination Module. However, the CorrectIllumination module has been producing images that separate and smooth out the wrong intensities.

I’ve attached screenshots of the original pre-processed images and the post-processed images that have been adjusted for brightness and intensity on ImageJ.

A) Pre-processed zoomed-out image

B) Post-processed zoomed-out image

C) Pre-processsed zoomed-in image

D) Post-processed zoomed-in image

So my questions are:

  1. Is the CorrectIllumination module the appropriate module to use to get the original pre-processed image to the level of intensity contrast of the post-processed image I included? If so, should I have the CorrectIllumination module on a separate pipeline from the one I attached below for efficiency?
  2. Are there any ways to isolate cells like A, B and C of the post-processed zoomed in image from false positives of similar intensities like the background surrounding those cells?
  3. In the pipeline I attached below, I have been using the Crop module to only focus on the area of the well instead of the four brighter corners like that of the zoomed out images?

The pipeline and a link to the original file of the pre-processed zoomed out image is attached below.
So sorry for the long message. Thanks for all the help!

SEGIL PIPELINE.cpproj (1.1 MB)