Dear Ahana,

As we have discussed off list your problems stem from differences in image quality and also a spatial miscalibration issue.

I just wanted to note a point that may be helpful for others. The registration software itself generates a "pyramid" of downsampled image stacks, first aligning coarsely with the most heavily downsampled image data and then more and more precisely. If you have imaged at high resolution (e.g. less that 0.5µm in any axis for a fly brain), you can tell it not to use the original image data using the --omit-original-data argument.

For example you can change your call from:

"/Applications/Fiji.app/bin/cmtk/munger" -b "/Applications/Fiji.app/bin/cmtk" -a -w -r 010203 -X 26 -C 8 -G 80 -R 4 -A '--accuracy 0.4' -W '--accuracy 0.4' -T 2 -s "/Users/ahana/Desktop/registrationfolder/refbrain/refbrain.nrrd/images" .

To:

"/Applications/Fiji.app/bin/cmtk/munger" -b "/Applications/Fiji.app/bin/cmtk" -a -w -r 010203 -X 26 -C 8 -G 80 -R 4 -A '--accuracy 0.4 --omit-original-data' -W '--accuracy 0.4 --omit-original-data' -T 2 -s "/Users/ahana/Desktop/registrationfolder/refbrain/refbrain.nrrd/images" .

Note that:

-A '--accuracy 0.4 --omit-original-data'

Passes additional arguments to the initial affine registration. And

-W '--accuracy 0.4 --omit-original-data'

Does the same for the second nonrigid (warping) step. There is rarely any reason to use full resolution image data for the affine step but there could be arguments for leaving it for the warp.

All the best,

Greg.