How to align two channels

This guide shows you how to correct a spatial offset between two channels.

Reference keys: preprocessing, alignment

Alignment protocol

  1. Launch the software. Open a project.

  2. Expand the PROCESSING block, scroll down to CHANNEL OFFSET CORRECTION.

  3. Click on the icon to set visually the shift values with a dedicated viewer.

  4. Set the overlay transparency slider to 0.

  5. Set the “reference” channel (Channel). Adjust the contrast to over saturate contours.

  6. Set the overlay transparency slider to 1.

  7. Select your overlay channel (the channel image to move relative to the first). Adjust the contrast to saturate contours.

  8. Set the overlay transparency slider to 0.5.

  9. Use keyboard arrows to move the overlay image and achieve perfect alignment in most places (see note). To achieve higher precision, fine-tune the shift values in the respective shift fields (and apply to view the result).

  10. Press set.

Note

A good alignment at the center does not guarantee a good alignment at the edges. This correction method only allows for 2D rigid translations, which may not be true for your data.

Apply correction

  1. Press Add correction.

  2. Once all preprocessing protocols have been defined (background correction, other channel alignments), press Submit. To apply the same protocol to all positions, accept the first popup.

  3. At the end of the preprocessing change the movie stack prefix in the experiment configuration to Corrected_.