I’m using Grid/Collection as part of a Jython script to stitch whole wells of multichannel images, some of which are easy to stitch (DAPI) and some of which don’t stitch well on their own (eg, small foci images). I had therefore taken the strategy of aligning the DAPI images via “normal” Grid stitching, then using the TileConfiguration.registered.txt file, changing the names in it (editing _DAPI.tif to _FITC.tif or _Cy3.tif in each line of the file names), and applying that alignment to all of the other channels using the “Positions from file” option.
This was all well and good, but on a recent batch of images I realized that while all my non-DAPI channels were always aligned perfectly, the DAPI channel itself (which I’d used to create the .registered file) was not always actually aligning to the other channels correctly, typically in the top-left corner of the image. Sometimes whole tiles were gone, other times they were off by 15-20 pixels. Sometimes it looked perfect!
Ultimately, I can work around this by then re-performing the DAPI stitching based on the .registered file, which worked well to “fix” all the stitches that worked poorly in my hands, but I’m worried it signals an underlying issue. Shouldn’t the positions in the .registered file match the stitch that is made in the same step?
Please let me know if you need follow up info/example images, or if I was unclear in any way.