Using Cell Profiler to detect Autophagy in Cells

I am worker at UCSD and we have been trying to use cell profiler to identify autophagy in cells. These spots are very small and I have been having only moderate success using a pipeline I made and was wondering if anyone has any tips or a better pipeline to use instead?

The biggest problem I’ve had is the variation in spots size and brightness/intensity. I can’t seem to find a good parameter for Cell Profiler to pick up these spots for a big sample size of images.

I have attached my pipeline and some sample images to the post. Hope someone can provide some advice and/or tips!

AutophagyValidationPipeline.cp (10.4 KB)


Unfortunately, since I am not familiar with the biology, I cannot tell from your pipeline where the inaccuracies lie, since it seems to be identifying a fair number of spots; perhaps an image showing false positives and negatives might be helpful. One key point to remember though, is that the filter size for EnhanceOrSuppessFeatures must be on the order of the size of the largest feature you want to enhance.