(part 2) Step 4: Registration

Now that all BB shapes are normalized,
we are going to register all volumes to each other
using the same procedure as in part 1, step 4.

Open in FIJI script editor a4_average.ijm macro.

It has an optional part for the registration of non-rescaled volumes that can be commented out.

The final average of averaged shape of NHS/total protein stain
was fitted again to find XY coordinates of the centerline using a4c_estimate_center.ijm macro.
These coordinates are used in the part3 (quantification) workflow.

input

The input are volumes in the a3_rescaling/rescaled folder generated at the previous step.

what happens

The macro will run AveragingND plugin,
that will make an iterative registration of average template (see explanation here).
In this case, we are not so much interested in the final averaged volume,
but rather in the final positioning/alignment of the volumes used for averaging.
They are the final result of this workflow.

output

The macro will create an a4_averaged/rescaled_avrg/ subfolder in the output folder.
The final average volume is stored there under the filename final_avg_N.tif,
where N is the best iteration.

The main output (registered/aligned volumes) is located
in the a4_averaged/rescaled_avrg/registered folder.

In case of extra averaging of non-scaled volumes,
a similar output is saved to a new a4_averaged/not_rescaled_avrg/ subfolder.