This is a long post, so I will try to organize it to make it easier to read. I am relatively new to confocal image analysis, but I have learned a lot through ImageJ tutorials. However I cannot seem to find the best way to associate cytoplasmic staining with nuclear masks.
My data: I have FFPE embedded tissue, stained with DAPI and various nuclear and cytoplasmic markers. I have tiled 40x confocal images (currently in 2D). I can make a nuclear mask to quantitate nuclear signals, however I am having trouble finding the best way to quantitate membrane markers, and keep their association with nuclear mask/nuclear ROIs. *** Beyond DAPI, none of the other markers will be positive in all cells. Weka seems like a popular tool (I haven’t really tried it yet), but not sure if this would be the best route for my data?
Ideal Analysis: I want to keep the X/Y coordinates of nuclei and associate positive cytoplasmic staining with corresponding nuclei. I don’t necessarily care to compare the intensity of cells/signal for this analysis, I just need the cytoplasmic values to be associated with the nuclear mask/ROIs and spatial coordinates (I can also choose the threshold for positivity in downstream analysis). Is there a way to analyze the outer perimeter of a nuclear mask within an x # pixel border?
- Dilating the nuclear mask is not good enough because too many nuclei become merged and inseparable with watershed.
- I have tried JACoP using nuclear mask with cytoplasmic mask, but I think this uses center based strategies which don’t seem to work for for my data either.
- Vornoi segmentation based on nuclei doesn’t work properly and always outputs a very strange segmentation.
Any tips/suggestions/strategies/plugins/key words to read up on are much appreciated! I keep going around in circles of ideas. Thanks in advance!!!