I have imaged the colon using 2-multiphoton microscopy (two channels, CFP and GFP). I repeated this microscopy experiments in three consecutive days, so I end up with three different Z-stacks of the same region.
For visualization purposes, I want to process the Z stacks the same way. The issue with in vivo Z-stacks is that the fist Zs are dim in intensity, then the intensity goes up the moment you go deeper, and at some point the intensity decreases again.
From the processing I would like to come up with Z-stack that are corrected for this uneven intensity. I would like to make it visible all the information of each image in the Z-stack, but I cannot just adjust the brightness of the lowest intensity slice because then some of my slices will become overexposed.
I would like also to process all the Z-stacks (from the different imaging sessions) for each channel the same way so when I present the data people see that all channels look with the same intensity no matter with imaging session I show.
First of all, I want to ask if it does make sense to normalize the intensities of each channel (e.g. GFP) from the three days all together. I know there are several options like Histogram Matcher but it does not work with IJ1 macro language and additionally you have to select manually the slice of reference if I am right.
I was wondering if someone knows which is the best option to do that. I am open to another ideas, I am amateur in imaging processing so I want to learn from more experience ImageJ people.