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Creating and aligning tracks

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.

Aligning tracks

Once your single tracks are completed, pinhole alignment is pretty easy.

  1. Quit LSM software
  2. Put the surface mirror slide on the stage and focus in its surface (in brightfield focus at the edge, then move to the middle.)
  3. Start PHadjust.exe
  4. Look at the single tracks it plans to align. Select or deselect as necessary.
  5. Press START.

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.

Creating Multi-tracking sets

Remembering your users multi-tracking settings, create each one from scratch using the (aligned) single track settings.

  1. Start LSM
  2. Click on Multi-tracking in Scan Configuration.
  3. Create a new track
  4. With that new track highlighted, click on the "Recording Settings" button next to the Track portion of the window. This opens a dialog box of all your previously aligned single tracks.
  5. Select a new multi-tracking setting from your list of single tracks.
  6. If you want a multi-track setting for GFP plus DAPI, select (for example) the "Alexa 488 (570).CH3," make a second new track, then select the "DAPI.CH2" setting.
  7. Resist changing the scan configuration "manually" as this can only cause pinhole alignment problems.

Scan Head configuration

Here is a diagram of our Zeiss 510 scan head filters

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.

Wed, Aug 10, 2005