Different cell positions depending on stain

I have a pipeline (attached) in which I process 3 images for a single cell sample, each image has a different stain: DAPI, GFP and mCherry. The pipeline does what I want, but there seems to be a slight shift in the image from the DAPI to the GFP and mCherry. I use the DAPI as the primary image to align to as the nucleus is nicely represented and the cell outlines are strong. I then use this as a mask to find the cells in the other images and calculate the amount of fluorescence in the nuclei and cytoplasm.

Is there a way correct for the image shift? Is this due to the use of different dyes and a shift in wavelength?
3 images may be viewed here: https://uwmadison.box.com/s/q7njoqvwh29tdba80m2waxa4meby31gq

Thank you,
Mike

masson_images_pipeline.cpproj (1.1 MB)

Hi,

There’s a module Align that can correct this shift, the default “Mutual Information” setting is already good for your case.

Please try. Good luck !