I am trying to quantitate foci by dividing total foci intensity/total nuclei intensity (not using per cell because the cells are very clumpy and haven’t been able to find settings that will separate all of them. However even when I set the threshold value high, I still get a value of 654 for intensity even though it says it doesn’t find any foci.



Hmm, I read this twice and still am not clear what you are asking. Can you explain what threshold you’re referring to? Better, can you post your pipeline and then refer to the appropriate modules when commenting?