Hi Kevin,

Looks like you have had a great progress.

The JBA can handle both local (non-linear) and global (linear) alignment problems but from my experience I will rely on other open source toolkit (ANTs/CMTK) for the global alignment.

Usually, we will create a pipeline for a bunch of similar images need to be aligned.

1) preprocessing step: I will process the subject image to make its orientation as the same to the template's due to most alignment software can not align images with bigger rotations especially having flips along the axis in some cases. Using some prior knowledge of the resolution info, we can make the subject image's scale the same to the template's.

2) global alignment to get affine matrix (you can merge previous step into this affine matrix as well)

3) local alignment to obtain the corresponding landmarks found in your subject image.

4) warp your subject image based on the transformations obtained from above 3 steps.

At Janelia, we develop a bunch of pipelines for different cases. If you need further help, please share your example image with us. I will find time to look into it.

Best,
Yang