Image alignment/registration

Correction of motion artefacts due to tissue contraction or other movement

The alignment workflow uses two plugins in Fiji: Linear Stack Alignment with SIFT and Template Matching and Slice Alignment. By default, they are run sequentially. The first one will correct for any warping in the image and the second plugin will reduce the lateral movement.

• Go to GAT -> Calcium imaging -> Align stack

• Click "Choose the file to be aligned"

• Click Open

• If the tissue is contracting and moving, then tick both options. However, if its only moving in XY without any 'warping' then untick first option. If you're not sure leave the options as they are.

• If the first option "Linear alignment with SIFT" was ticked, you'll get another image stack which will have the name "Aligned" at the start. The alignment processmay take a while depending on the image size. We recommend trying it on a small substack to test if its working.

• If the Template matching option was ticked, it will undergo a second round of alignment to correct any lateral movements.

• The final image will be the aligned dataset. A copy of this will also be saved in the same folder as your original dataset.