I’ve outlined my underlying data set in a separate post. I am now looking to take the same data and create a “gold standard” for comparison. The idea is to make a MIP image of the red (dead cells) and green (live cells) channels and then count them manually, with an additional channel merge image to clarify which cells appear alive on the green channel but are actually dead/dying by looking for colocation of the red channel.
Regarding pixel intensity. I have problems with my z stacks on higher cellular density images. That is the confocal wasn’t able to penetrate as well deeper into the stack. However, when I “window” the image well enough I can in fact see everything. So, my question is does this matter? If my understanding of MIP is correct, it doesn’t matter how terrible the penetration is - if the cell is even just barely visible it should show up well on the MIP. Am I missing something?