Dear QuPath users,
I have several consecutive sections that have been stained with H-DAB for two different antigens.
I am trying to align these sections, so that I can look at co-localization.
For this I have tried two things:
- Qupath Image Overlay Alignment > this gives me great alignment, but the aligned images cannot be exported, ‘saved’ or used otherwise
- Exporting the image to Image J and use something like HyperstackReg (first deconvolute the H and DAB color channels, then register the images using the hematoxylin and then stack the images to obtain a ‘double-staining’ or immunofluorescence-like image.
While the second approach works, I find that the alignment shown in the example window in Qupath is very much superior to Hyperstackreg.
I then tried a third approach, where I copy the Affine matrix (a 3x2 matrix) and use this matrix in TransformJ (which asks for a 4x4 matrix). I just copy the matrix that I get from QuPath and leave the rest as is.
0.9360035061836243 -0.24853743612766266 1485.8318112182617 0 0.24227723479270935 0.934749960899353 990.21954647827150 0 0 0 1 0 0 0 0 1
If I apply this transformation to the image this looks okay visually, but when I stack the images together in a Stack using the “Images to Stack” using “Copy (center)” in ImageJ there is still a clear mismatch.
Do you have a suggestion on how to use the transformation matrix that QuPath outputs in an external program? Or is this means of stacking the images the problem?
I feel it would be a shame to use an ‘inferior’ approach of alignment.