Posted by
Kenneth Sloan-2 on
Feb 24, 2019; 4:33pm
URL: http://imagej.273.s1.nabble.com/Hyperplexing-multi-channel-immunofluorescence-images-tp5021831p5021833.html
Everything depends on the details, but...
I would start by deciding what flavor of transformation you
actually need. I would avoid even thinking about local warping
until and unless you find it is absolutely necessary.
Assuming that the tiling done by the Zeiss slide scanner is
adequate, I would create a low resolution overview image for
each staining step and align these.
Sorry - I don't know how to communicate the resulting transformations
to the full .czi pyramids.
Without knowing what you really need to do, I might also consider
processing the 11-deep stacks tile-by-tile.
If you NEED the full image, then perhaps lower resolution is adequate, and
you don't need a full pyramid. On the other hand, if you need the finest
available resolution, processing tile-by-tile may allow you to compensate
for more complicated warping of the tissue (each tile might be adequately
registered using a simple transformation.
Finally, you might do the high-resolution work on the individual
(smaller) stacks, and later register a higher-level, more abstract
representation of the results.
I don't personally use TrakEM, but I have colleagues who swear by it.
In a somewhat similar situation (serial section and block-face EM),
they produce .obj models of what they see at high resolution, and
then ask me to register and combine the .obj models.
This works surprisingly well.
Above all - it's important to have a model of the CAUSES for
mis-alignment. Is it imaging? tiling? tissue warping? Different
causes call for different fixes.
--
Kenneth Sloan
[hidden email]
Vision is the art of seeing what is invisible to others.
> On Feb 24, 2019, at 07:21, Doc Watson <
[hidden email]> wrote:
>
> My apologies if this has been asked before, but I wasn't able to find
> previous threads that really answered this. Here's what I'm trying to do:
>
> I have tissue sections imaged on a Zeiss slide scanner that produces large,
> tiled and stiched, pyramid composites with 20x magnification, and 5
> fluorescent channels (.czi format). Then I'm doing pretty standard
> cyclic-IF where I strip the fluorphores from the tissue, stain with new
> markers, and image again. This leaves me with 8-10 5-channels images of the
> same tissue that I then want to align and merge together to create one image
> with ~40 channels.
>
> So I need to align every channel from each staining round to the images from
> the first round of staining, and probably perform small local deformations
> in case the tissue is warped during the stainings. Ideally, I'd like to use
> the DAPI channel from each 5-channel image to perform alignment and
> registration, so that any image deformation is also applied to the other
> channels from that round of staining.
>
> I'm looking at using TrakEM2, but does anyone have other suggestions for how
> to approach this?
>
> And does anyone know a way to keep the pyramid structure of the .czi file,
> or will I have to convert the whole thing to a BigTIFF image?
>
> Thanks!
>
>
>
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