Vox

Setting up the Nikon Perfect Focus System (PFS) for multi-point acquisition in Z

The following is a procedure I've worked out for setting up the PFS for multi-point acquisition. I used fluorescent HeLa cells grown on Mat-Tek glass-bottomed 35 mm dishes and mounted in a Solent Scientific insert designed for a range of 35 mm dishes but adjusted to hold this one firmly in place. I cannot say for sure that the procedures will work for all surfaces. Nikon only recommend glass of standard #1.5 coverslip thickness (170 µm).

1. Make sure that the PFS lever under the microscope stage on the right is in the In position - otherwise the PFS won't work
2. Focus on the specimen. I focused on the cytoplasm of well spread cells so that my focus plane was relatively near the coverslip
3. The small green LED on the front of the microscope stand should be lit, indicating that the coverslip is in range of the PFS
4. If it is not lit then slowly lower the microscope focus until the LED comes on. Note that the LED may only be on in a very narrow range of focuses, so don't focus too fast or you might miss it
5. Focus up and down until you are about in the middle of the range of focus where the LED is on
6. Press the Focus button on the front of the microscope to turn on the PFS
7. Use the Offset adjustment dial to the right of the microscope stand to refocus on the focus plane
8. Bring up a live image of the plane of focus in the Vox image preview
9. Use the Offset dial to readjust the focus on the screen, then turn off the Focus button
10. Start marking points, making sure at each point the small green LED on the front of the microscope is on
11. If the LED is not on or flickers at any of the points, adjust the microscope focus until the LED is solidly on, then mark the point
12. After all points are marked, go back to the first point and turn on the PFS to make sure the focus has not drifted. Make any fine adjustments to the offset if necessary
13. Turn off the PFS and set the Ultraview Focus Drive to Zero
14. Set the Top and Bottom of the Z series. Note that because zero is at a plane of focus near to the coverslip, the top of the Z series may be further away from zero than the bottom (e.g. Bottom = -5.0 µm, Top = +25 µm)
15. Review the points to make sure the LED is still on at all of them
16. Check the Bottom and Top of the Z series settings at a few points to make sure that the focus range is adequate for all of them. Make adjustments if necessary
17. Go back to the first point
18. Set up the time-lapse settings, setting the autofocus method to 'Nikon Ti PFS'
19. You can now either turn the PFS on before starting the time-lapse or leave it off. If you choose the former this should keep the PFS on between time-points, but this might not be necessary if your time-points are close together.

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