Finding a huge ratio difference between these is gonna be tricky, because your segmentation is never gonna be perfect based on how clumpy your cells are (but hopefully could be improved with some tinkering), you’re trying to hit a very fine region in the PM, you have the out-of-focus light as @Minh mentioned, and some of your cells (at least in the either seem not to have the Cy5 labeled protein at all or a whole clump will only have it at the exterior rather than at every edge). You can see what I mean in the image below (outlines are cell outlines, with Ratio1 values overlaid)- the cells with a pretty good segmentation have pretty good ratios, but a lot of the ones with no protein or bad segmentation don’t. You could potentially try to come up with some filters to try to more clearly get the cells you wanted, though it might be tricky. Definitely it’s worth trying to clean the segmentation, though I’m not sure how much better it can get due to the clumping.
I think if you swap though from trying to look at the absolute average difference at the plasma membrane to looking for what percent of cells show at least a moderate plasma membrane enrichment, the differences might be more apparent; if I use a (somewhat arbitrary based on the image I showed) threshold of your “Ratio1” of 1.1 as a threshold for enrichment (after switching to measuring in Crop rather than Corr), I get 58.7% of cells showing PM enrichment in the first image and 16% in the second, which to me shows a real difference. I’ve attached the edited pipeline here. PM-cytoplasm_BC.cppipe (21.6 KB)
As for the Vitra, remember that Cell = Nucleus + Cytoplasm, so the best measure of translocation is going to be Nucleus/Cytoplasm (or Cyt/Nuc), not Nuclei/Cell or Cytoplasm/Cell. If you do that, just from the back of the envelope you posted here, you’d end up with (.197/.136)=1.45 for image 1 and (0.206/0.175)=1.18 in image 2, which is a much bigger change.