Z offset, x and y shift, Zeiss axio imager

Hello everyone,

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!


I suspect most people use some variant (not advocating for any particular brand) of a submicron tetraspeck-like bead with multiple channels of fluorescence excitation and emission, and measure the offsets between channels and apply them. Only thing to keep in mind there is to use the center of the beads, since you’ll have a larger point spread function/spot size with the longer wavelength.

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sorry for the late reply, thank you very much for your answer!

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