(Streamlining from the forum thread) - I just figured it would be better to move it here.
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. I know that theoretically Grid/Collection can be used on multichannel data, but in my experience that had issues and this worked much more smoothly.
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 it looked perfect, sometimes whole tiles were gone (which @imagejan pointed out was probably due to #35) , at the most concerning times they were off by 15-20 pixels.
Screenshots below - green is the nuclei as output by the first run of Grid/Collection stitching, red is the stitch I got by stitching from the TileConfiguration, teal is phalloidin). I'm most concerned about the cases with offset- I can make my DAPI channel match by going back and re-stitching it according to the .registered file at the end, so I have a workaround, but if something is off I figured you'd want to know.
(Streamlining from the forum thread) - I just figured it would be better to move it here.
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. I know that theoretically Grid/Collection can be used on multichannel data, but in my experience that had issues and this worked much more smoothly.
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 it looked perfect, sometimes whole tiles were gone (which @imagejan pointed out was probably due to #35) , at the most concerning times they were off by 15-20 pixels.
Screenshots below - green is the nuclei as output by the first run of Grid/Collection stitching, red is the stitch I got by stitching from the TileConfiguration, teal is phalloidin). I'm most concerned about the cases with offset- I can make my DAPI channel match by going back and re-stitching it according to the .registered file at the end, so I have a workaround, but if something is off I figured you'd want to know.
LMK if I can provide any additional info!