If fluorophores are not only visible in one channel but ‘bleed through’ into the next channel they can be linearly unmixed using this macro, as long as at least some pixel are ‘pure’ for each fluorophore (only visible in this channel).
- Input a stack of 2 or 3 slices.
- ROI select a background area, then run 'get background' macro.
- ROI select an area with pure fluorophore1, then run 'get fluo1' macro.
- ROI select an area with pure fluorophore2, then run 'get fluo2' macro.
- run 'unmix' macro.
- The output are 2 pictures named 'channel1' and 'channel2'.
- Parameters are kept and used from a parameters image.