Zeiss multi-tracking is an indespensible method of acquiring images of samples probed with more than one fluorophore. However, creating and calibrating the individual tracks of a multi-tracking setup can be a bit problematic. There is one concept that you should remember: Always create individual tracks in Single Track mode ("Recording Settings") and copy those tracks when creating Multi-track settings.
Making a single-track setting for every possible multi-track setup can be a difficult at first due to the myriad possibilities of channels, filters and lasers. However, after noticing the habits of your users, you should be able to come up with the top 5 or 6 multi-track setups. Work backwards from these and come up with the single track settings that the multi-tracks are built upon. Name your single tracks with descriptive names such as "Alexa 488 (570).CH3." Then use these single tracks UNMODIFIED when building multi-track settings.
Once your single tracks are completed, pinhole alignment is pretty easy.
PHadjust will start the laser(s), and go through the alignment procedure for each single track selected. This can take a while if you have a lot of tracks.
Remembering your users multi-tracking settings, create each one from scratch using the (aligned) single track settings.
The Zeiss 510 in the Biological Imaging Facility has been setup with the following Multi-Tracking sets. These cover most of the most common fluorophores.