Aligning 4 rotated 16 bit images


I have 4 16 bit images that I took by rotating a sample 0, 90, 180, and 270 degrees respectively. Since I rotated the sample manually the fields of view in the images don’t line up perfectly. I’ve been trying align them in order to then make a composite image. I’ve tried Big Warp, SIFT align, and Align_RGB_planes so far with no luck. Also a side note, I’ve rotated all the images back to “0” degrees before applying these methods. I don’t want to end up with a stack of images, I only want one 2D image at the end if that helps. My images are of mouse skin (collagen is visible) with second harmonic generation microscopy if that’s important information.

I’ve included the already rotated images below.
ChanA_001_001_001_001.tif (8.0 MB)
ChanA_001_001_001_001-1.tif (8.0 MB) ChanA_001_001_001_001-2.tif (8.0 MB) ChanA_001_001_001_001-3.tif (8.0 MB)

Hi @breanna.collins,

Welcome to the forum.

Sorry things haven’t worked for you yet…what was wrong with the results you’ve been able to get so far?

I used Bigwarp to generate this alignment below in a minute or so.
Is something like this acceptable? If not, why?

Hope this helps

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The result you got with Bigwarp looks perfect for what I want. So it seems I’m just not very proficient at using Bigwarp, I’m a bit of a noob. I’ve looked around on this forum and online and I can’t quite figure out how you got that image. I’ve gotten to the part where I’ve made landmarks using 2 of the images but I can’t figure out how to do anything past that. The video tutorial with no sound that’s floating around has not been very helpful past this point.

When I go file>Export as ImagePlus> it gives me an image of pure white with the Left and bottom edges of the image appearing all black. Do you know of any other resources? Thanks for all the help so far!

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Glad you thought to post on the forum!

After placing some landmarks, I pressed T to “apply” the transformation in bigwarp.

Below are the points I clicked - if you save that as a csv file and load them into bigwarp
with File > Import landmarks.

For my example I used:
Moving image : ChanA_001_001_001_001-2.tif
Fixed image: ChanA_001_001_001_001.tif

Exporting may be a little tricky for you since the bounding box of the moving image isn’t the same as the target image. I’ll write more on that in another post

Hope that helps, and good luck


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Thanks so much for the help! I figured out that I just needed to adjust the contrast, but the above stuff really helped. The default settings made any part of the image appear only white while the places where the image didn’t overlap appeared black.

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