Qupath image overlay/ alignment analysis

Hello everyone, how can I do multiple IHC analyses by image alignment on QuPath? If I have one slide each for CD3, CD20, PD-1, and CD68, can I generate one image by overlapping it on each other?

In theory, yes, there are a few posts about that on the forum. You can either segment objects on one and move them between images, or convert color deconvolved channels from each image into a single image.

In practice, it depends on what kind of data you want to get out of them. Sequential slices are nearly useless for cell-cell colocalization (the same cells just don’t exist on sequential slices often enough), but can be used over broad areas like tumor vs stroma. Strip-stain can allow for finer-grain analysis, but the affine alignment in QuPath is not generally going to be good enough for double/triple positive cells.

Even the deformable alignment in Visiopharm has usually failed to get accurate alignment on the cellular level, but can allow for better gross alignment of sequential slices for general region analysis.

Thank you for your response. Sequential slices create problems with alignment.