CellProfiler Difficulty Identifying cells on some slides with high background

Hello, I have been struggling to make an IdentifyPrimaryObjects that identifies both the cells within the darker background and within the brighter part of the slide. I have tried multiple things - I assumed that the adaptive thresholding would work, but it seems to still miss the cells in the bright areas. I was wondering if anyone had any thoughts? 01Plate_withDrug_05Day_02-Image Export-101_Rhoda_ORG_Overlay.tiff (4.1 MB) 01Plate_withDrug_05Day_01-Image Export-100_Rhoda_ORG.tif (2.8 MB)

Hi @RussellH,

It looks like the background in the central areas is particularly strong, it might be worth trying to do some background subtraction or illumination correction before attempting to detect objects. We might be able to provide further advice if you’re able to upload a copy of the pipeline you have so far.

Hope that helps

Hi @DStirling,
Thanks for getting back. I am not sure how I would go about doing that. I have uploaded the pipeline I used. RECA_3Testing.cpproj (1.5 MB)

Hi @RussellH,

Looking at your pipeline, there are a number of ways you might want to approach this. You might be able to just use the EnhanceOrSuppressFeatures module to pull out the smaller objects for detection, I’d give that a try first. If that doesn’t work perhaps try the CorrectIllumination modules in ‘background’ mode.

Thank you @DStirling, I will try those things