Hi All,
I was using the plugin for 10X images of neurospheres and ran into issues with alignment. I took images in a snake pattern with a lot of overlap (>50%). I then used the "unknown position" option for the Grid/Collection stitching; I got better luck with "unknown position" than with "Grid: snake by rows". The result is mostly accurate, but the nuclei from overlapping constituent images are juxtaposed in the fused image instead of merged together (please see attached).
I've tried to increase the regression threshold and decrease the displacement threshold, but without any luck. Are there any other settings I can tweak?
Thank you for your help.
Sincerely,
Cheuk
Have you tried doing this pairwise to build up the image sets?
http://fiji.sc/Image_Stitching
Hi Dr. Morris,
Thank you for your suggestion. I've tried using pairwise stitching but even that sometimes could not properly align the images. A kind ImageJ developer pointed out that the amount of overlap needs to be very consistent among all images to obtain optimal stitching. I was able to get a good fused image on one of the fluorescent channels (Tuj1), but the alignment is faulty for the Hoechst channel when I use stitching on the Tuj1/Hoechst merged images.
I was trying to reuse the coordinates generated from the Tuj1 alignments, but I get an error saying the plugin could not find the file even though I put the TileConfiguration file in the same folder as the images:
ERROR: TextFileAccess.openFileRead(): java.io.FileNotFoundException: J:\current_labmembers\Cheuk\Scope Pictures\Neurite outgrowth assay 1\08C6 24hr Grid\A\Overlay\08C6 24hr_A Tuj1 10x.registered (The system cannot find the file specified)
ERROR: Stitching_Grid.getLayoutFromFile: Cannot find tileconfiguration file 'J:\current_labmembers\Cheuk\Scope Pictures\Neurite outgrowth assay 1\08C6 24hr Grid\A\Overlay\08C6 24hr_A Tuj1 10x.registered'
ERROR: Error during tile discovery, or invalid grid type. Aborting.
Is there something I can tweak in the "position from file" option? I'm also trying to retake some pictures to have more consistent overlap.
Thank you for your help.
This is something that is best handled on the fiji development list. It sounds like you are already in communication with them. As I'm not the developer of the stitching plugin I think it's best if you refer to that person.
Hi Dr. Morris,
Thank you for your response. The pairwise stitching will be a good alternative for smaller pictures, and I'll have to keep working on image capturing and reusing coordinates. Thank you again for your help (I thought I'd sent a message out earlier but I guess it got lost in the shuffle).
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