Posted by
CARL Philippe (LBP) on
Oct 08, 2019; 7:05pm
URL: http://imagej.273.s1.nabble.com/Multi-stack-registration-of-single-channel-confocal-images-tp5022499p5022501.html
Dear William,
Please look at the following discussion:
http://imagej.1557.x6.nabble.com/registering-a-stack-advice-needed-td5020546.htmlMy best regards,
Philippe
Philippe CARL
Laboratoire de Bioimagerie et Pathologies
UMR 7021 CNRS - Université de Strasbourg
Faculté de Pharmacie
74 route du Rhin
67401 ILLKIRCH
Tel : +33(0)3 68 85 41 84
----- Mail original -----
De: "wmacturk" <
[hidden email]>
À: "imagej" <
[hidden email]>
Envoyé: Mardi 8 Octobre 2019 19:29:35
Objet: Multi-stack registration of single-channel confocal images
Hi all,
I recently took some confocal Z-stacks of the exact same slide and XY
position in two separate channels (i.e. I found my ROI in the slide, took a
Z-stack with the 488 laser, then switched to the 405 laser and took the same
Z stack). The fluorophores were DAPI and eGFP, and I was concerned there
would be some fluorophore coupling -- the DAPI emission overlaps with the
eGFP excitation -- so I did the eGFP first, saved that stack, then ran the
DAPI and saved it to a different file. There's some XY drift in the sample
for whatever reason, with the DAPI stack obviously pushed to the right by
quite a bit, and even within the stacks, there is some drift as you traverse
the Z axis. Using stackreg it's no big deal to align the stacks to
themselves, but when using multistackreg, the result is still not aligned. I
believe the problem is that the eGFP stained bright everywhere except the
chromatin, and the DAPI stained in the opposite way. One of the cells seemed
to be undergoing mitosis, so the green channel shows a big green blob with a
kind of hole in the middle, while DAPI shows just a blue blob in that hole.
When running stackreg on the blue channel, most slices line up, but in some
focal planes, the plugin thinks one of the chromatin bundles corresponds to
a different bundle from a previous slice, so those slices get misaligned (by
quite a bit). I can manually go in and shift slices to roughly the right XY
position with Translate, but that's tedious and won't work when I start
using bigger stacks.
I'm using FIJI, and would like to automate this process fully. I'm sorry if
the question is vague, but can anybody help out? Happy to provide examples
and further info as needed.
V/R
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