I'm a very new user of CellProfiler and I'm having trouble figuring out a strategy for segmenting cell membranes labeled with a genetically encoded (i.e. not externally stained) fluorophore. I have high-mag images which contain a few cells labeled with:
BFP-H2B (very tightly confined to nucleus)
RFP-FarnesylationTag (shows significant enrichment on cell membrane, but there is weak cytosolic fluorescence and seems to clump in the ER as well)
GFP-ProteinX (protein of interest tagged with GFP).
I am interested in tracking how well ProteinX localizes to the membrane, which I think is conditional on a certain stimulus. I can identify cells using IdentifyPrimary on the nuclei, and I was then planning to use the RFP image as a guide for co-localization to see where the membrane is, and then see what fraction of the GFP image is within that mask. However, the localization of RFP to the membrane isn't perfect, and I don't want the cytoplasm and ER to be inlcuded in the mask. I can get rid of the cytoplasm by thresholding. Is there some sort of topology-based filter that I could use for eliminating the internal cluster at the ER?
I've uploaded the image files:
Outputs.zip (928.8 KB)
Thanks for any help!