Apologies if this question has been addressed before. I hunted around the forum, but wasn’t able to find answers to this specific problem.
I’m trying to combine 8 different 5-channel fluorescent images of the same tissue section to create one 40-channel image. I’m doing fairly standard cyclic-immunofluorescene where I stain, image, remove the fluorphores, and repeat. I’m imaging on a Zeiss Axioscan slide scanner, which gives me large tiled and stiched images with 5 fluorescent channels. The images are usually shifted in X-Y in each round of imaging , but there may be small changes in rotation as well (and I can’t rule out small deformations in the tissue between rounds of staining).
Ideally I’d like to get perfect cell-to-cell aligment across each round of staining for every channel.
The channels of each imaging round seem well aligned to each other, so what I’d like to do is use the DAPI channel of each 5-channel image to do the alignment (that way any changes are applied to the other 4-channels of each image).
I’ve tried combining low-res versions of the tiled images with a few of the included Fiji registration plugins, but the process either fails, doesn’t allow me to choose which channel to use for aligment, or doesn’t include all the channels in the final output image.
This might be a fiarly routine issue, but I just don’t know ImageJ well enough to solve it. Does anyone have any thoughts or advice on how to proceed?