I’m trying to create an automated CellProfiler pipeline that can successfully identify, segment, and count cells. I have been struggling to get a pipeline and set of parameters that works consistently, which I believe is due to background variation between images. I have tried illumination correction, enhancing speckles, closing, and a median filter. Here is the pipeline for reference: Method_2.cppipe (37.3 KB).
This works decently for images that have a darker background:
Original image: RAT1_VMS2R_RESULTS_CH3.tiff (675.3 KB)
But when there is a lighter background, the brighter background regions are recognized as objects as well:
Original image: RAT1_VMS2R_RESULTS_CH2.tiff (675.3 KB)
Across my image set, there are both kinds of backgrounds. I was hoping to devise a pipeline that could work consistently for both. Any advice for anything more I can do to correct for this? If not, do you have any suggestions for other software that might be able to handle this better?