I am trying to create a pipeline for the analysis of neurons.
I have 4 channels:
-Channel 1 is nuclei and I defined them as primary objects
-Channel 2 is subtype 1 of neurons and I defined them as secondary objects (secondary object 1)
-Channel 3 is subtype 2 of neurons and I defined them as secondary objects as well (secondary object 2)
Now I want to measure the intensity of channel 4 in both the secondary object 1 and 2 but it seems like the intensities are the same (one should give me 0), do you know what could be the problem or how I can specifically measure the intensity only in the subtypes and not in all cells?