Posted by
CARL Philippe (LBP) on
Oct 09, 2019; 9:06pm
URL: http://imagej.273.s1.nabble.com/Multi-stack-registration-of-single-channel-confocal-images-tp5022499p5022512.html
Dear William,
The advice I wanted to give you is that in the case the stagreg plugin is almost doing the job you are looking for, than you should give an alterative try to both the swift and template matching plugins.
Nevertheless, if I would be walking in your shoes (and I apologize very sincerely in advance if you think that what is going to follow is rude, but given that I'm french it is quite in my nature, isnt it? Thus, I hope you may forgive me at least a bit!) I would just take your data and simply trash them!!!
Once done, I would sit back on the microscope and acquire new pictures but not following the workflow you described but an alternative one...
Indeed, if you want to do something "clean", you should rather acquire the different channels as childs of your Z-stack to actually be sure not to get the drift effects you are reporting.
Also I suppose that you are not using an air objective, but rather an imersion (oil, silicon, glycerol,...) obective.
And given that these imersion media have a quite high viscosity, it is better to make your Z-stack acquisition from top down rather than from bottom up (and if you make multiposition acquisitions, adding waiting time for the imersion media to stabilize may be needed).
Respecting these rules I make the bet that your new acquired picture won't anymore need the realignments you reported.
Good luck and make our planet great again!
My best regards,
Philippe
PS: As I'm writing this answer I have a 24 hours (acquisition every 10 min) > 17 x-y positions > 5 heights Z-stack > 3 channels acquisition of a wound healing running...
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: Mercredi 9 Octobre 2019 02:37:25
Objet: Re: Multi-stack registration of single-channel confocal images
CARL Philippe (LBP) wrote
> Dear William,
> Please look at the following discussion:
>
http://imagej.1557.x6.nabble.com/registering-a-stack-advice-needed-td5020546.htmlThanks for the reference Philippe. Unless I'm missing something, though, the
discussion doesn't seem to address my issue. I believe the main problem is
that, as these are confocal images, and the nucleolus is not a perfect
sphere (rather oblong, and imaged at an angle), the top slice shows a
portion that is noticeably up-and-right from the bottom slice, with no other
landmarks for registration. I'll attach two slices to show what I mean.
<
http://imagej.1557.x6.nabble.com/file/t382446/DAPI_top.jpg>
<
http://imagej.1557.x6.nabble.com/file/t382446/DAPI_bottom.jpg>
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