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Note that Perkin Elmer don't recommend having the PFS on between time-points, but if there is a lot of focus drift between time-points it may be the only way to stop the focus drifting out of the range of the PFS. Here are some links:
- Perkin Elmer Tech Note - Nikon Ti PFS - PE's technical note on using the PFS. The last section, 'Multiple planes with secondary drive (piezo), multipoint, over time' deals with the above case. My method differs in that I make sure the PFS is in range initially by adjusting the microscope focus to the middle of the range where the LED is on.
- Austin Blanco's blog on PFS troubleshooting - Some useful information on the operation of the PFS, ideal specimens and error indicators