I do multiplexe immunofluorescence and I use zeiss axio imager for imaging my slides. I noticed that when I set the focus on DAPI channel, the focus in other channels (FITC, cy3 and cy5) is not as sharp as in DAPI channel, especially in the red spectrum. I believe this is something known and one way to work around it is to set a Z offset to correct for this small difference between channels. I would be grateful to know what is the best way to set the correct Z offset between channels? I for now use the Z stack option and I just set the right focus of DAPI channel, set it as the first point of the Z stack, then move to cy5 channel and adjust the focus, then mark it as last point of the Z stack, then I check how much distance (in um) between first and last point of Z stack, and this is what I set as Z offset. I am not sure this is the right way??
Related to this, I see that there is two more options next to Z offset: x and y shift (in px). what are these options for? are they necessary to use and in which situation?
Thank you very much for your input!