Struggling trying to align images using ImageJ


I am struggling trying to align several images using ImageJ. The images are the following:

  • 4 images of the same spot in the tissue that belong to 4 different channels (blue, yellow, magenta and green)
  • 2 images of the same spot but at different time point. In this case the staining for yellow is the same but green is different (magenta and blue are missing).

I would like to align these 6 images together based on the alignment of the yellow channels since they are the same. The rest of the channels have different staining therefore I cannot align all the 6 images together (or at least I have not been able to do so).
Can someone give me an approach to accomplish this task? Sorry for this potential naive question.



Good day Jorge,

without seeing typical images it appears rather difficult to help, especially because you don’t tell us about the kind of alignment, pure translations, or size, rotations, etc. as well.

Yes, it makes a big difference!



Hello @gerg3x and welcome to the ImageJ Forum!

Can you upload some images and explain here what plugins have you tried?

Hello! Thanks for the reply. Find attached the 6 images I want to align. There are 4 images that are the four channels used in the scanning (33A-B / 33A-F / 33A-M / 33A - R) and then 2 images from the same spot at different time point (33C - F / 33C - R).

33C - R and 33A - R have the same staining (endothelial cells) and the rest are different. I want to use these two images to align the rest and create a final picture to observe colocalisation. I can align the four images of 33A and the two images of 33-C separately since they have the same field of view but my idea was to merge all of them using the endothelial cell staining.

I have used the plugging Linear stack alignment with SIFT but they align all the images together (I’m starting using ImageJ so I don’t know many pluggins).

Thank you.

The other image missing: Img33C-000211-R|nullxnull

Hello @gerg3x

I’m a bit lost here, which images exactly do you want to align to which images?

Isn’t that what you want? To have all images aligned in the same space?

Hi iarganda,

Linear stack alignment with SIFT align all the images in the stack which means that only the ones with the same staining will be aligned. I want to create a z image with all the images.


I tied to align your images. Is this OK?


First, I combine these image to one multi tiff. (4 channels, and 2 times)
And then, I align the image own plugin that can align Hyperstack image.
*I use around a bright spot on “33A-F and 33C-F” images.


Hi hwada,

Thank you for the alignment. However, I can only see the nuclei in the picture and not the rest of the stainings. I followed more or less the same approach. I combined the images in two multi tiff images and then align them using the linear stack alignment with SIFT. In the end I have a stack with the 2 images aligned but I would like to combine these 2 images of the stack into a final one.



The uploaded image is a multi-tiff containing all your images.
If you did not download, please download it and open the image by ImageJ.
And then, please convert stack image to HyperStack image.(4channels x 2times)
*If you need to see composite image, image type should be changed.
After that, adjust the brightness of each channel.

*I upload a small scale image as gif animation image.



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Thanks a lot for your help.