Z-stacking of a multichannel image

Hi! I’m working with tissue sections stained with several fluorophores. Some of these channels are in focus at different z positions, so I’ve taken MDA images with z-stacking. In the resulting composite I know how to flip between channels and adjust their contrast if necessary. However, how can I select the appropriate z stack for each channel? (Changing the z-stack on the slidebar affects all the channels). Thanks!

Hi @cmped,

I am not entirely sure if I have understood your issue.

That is intended behavior since it doesn’t make too much sense to e.g. show different z-slices from your multi-channel images as a composite or merge.

Best,
Stefan

Hi @cmped,

What is MDA?

You could split the channels up in to separate z-stacks and then focus each one separately, find the offset and add slices to the beginning/end of the channel stacks where necessary to bring them in to alignment. But this makes a bias as to the colocalisation of the labelled structures, you assume they are sat in the same focal plane. If you’re measuring this, then you shouldn’t align the channels this way, you would need a marker (e.g. fluorescent bead) that is present in all channels.

The problems may be occurring because your microscope has an objective lens that doesn’t correct chromatic aberration.

Rob