I am new to CellProfiler, but have a lot of experience with FIJI. I modified one of the default pipelines to help me assess cytosolic signal vs nuclear signal of the protein stained in channel 2. I used channel1 (DAPI) to segment the nuclei and channel 3 (actin-don’t judge I didn’t prep the samples and the lab that did likely had outdated Phalloidin :-)) to segment the cell. My goal is to measure the nuclear and cytoplasmic mean intensity of channel 2, ultimately reporting a ratio of cyto:nuc. I thought my pipeline was working, but I appear to have layers/ghosting from previous samples on my images that are used as input to the object analysis. It seems the input image isn’t reset when going to a new image, I noticed this by getting more and more DAPI as I processed more images. DAPI signal that isn’t present in the original image not processed through cell profiler. Do I need to clear the objects, etc at the bottom of my pipeline somehow?
Attached is a screen shot using FIJI showing two images that were processed through Cellprofiler…top row processed first, bottom row processed 2nd. Second row shows residual nuclei from the first image.
cytoplasm_nuclear_ratio3.cpproj (141.2 KB)