Sorry, I’m sure its been asked but I couldn’t find it through the search function but are Z stacks mandatory for multiplexed images registration and if so how does one ensure that you capture the same focal plane with each cycle? Is there a rough guideline for optimal stack size (I see 1um in some articles) and slice interval?
This question seems to be missing massive amount of context.
ITK can register 2D images, and various kinds of 3D images. Best for experimentation is 3D Slicer, via its extensions elastix and ANTs. I advise to try registering your images using GUI, before writing any code.
Hello @PART,
Z stacks are not mandatory for multiplexed image registration. When working with a 2D image there is the implicit assumption that you captured the same focal plane in each cycle or that you are close enough (epsilon for close enough is dependent on your requirements).
When working with a Z stack this is easier because it is more likely that there is spatial overlap between the images. It is still possible to have two Z stacks without spatial overlap if you don’t image the whole physical sample, but that is more of a user error in image acquisition, so rare.
Relevant code/programs for multiplex affine registration using a common channel:
- SimpleITK Imaris Extensions, specifically the XTRegisterSameChannel. This extension has accompanying data and a video illustrating how to use it.
- The above extension is built upon the sitk-ibex code.
If you have questions about those programs, please ask on their respective GitHub issues (SimpleITK Imaris Extensions issues and sitk-ibex issues).
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