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  1. Start the microscope as usual, but without turning on the software

  2. Turn on the EPI fluorescence lightpath and focus on your sample using the ocular (e.g. 1 μm TetraSpeck beads)

  3. Adjust the lens correction collar for the immersion medium, the cover glass thickness and temperature you are using, if the lens has one before turning on the ZDC (not all lenses are compatible with ZDC, for list see below)

  4. To adjust the Near Limit Setting for the objective lens you are using go to the Settings menu by pressing the spanner icon on the touch panel controller

  5. Select Customized -> Focus Limit Setting

  6. Look down the eyepiece and focus slightly above your sample

  7. To set the Z-position you are now in as Z-Focus Limit press Set and then OK on the touch panel controller

  8. Close the Settings menu

  9. Make sure that the Z-drift compensation port switching lever (on the left under the stage) is in the IN position. This engages the dichroic mirror in the light path of the Z-drift compensation sensor unit and therefore enables Z-drift correction



  10. Check that the DIC slider (on the right under the objective turret) is removed from the lightpath (pull out)

  11. Open the FV10-ASW 4.2 acquisition software

  12. Setup the light path, start a live preview and check the sample is in focus

  13. Make sure to set up a time series as ZDC is only available in the time series mode

  14. Click ZDC -> ZDC Settings in the software



  15. Go to the live image preview again and focus near the glass coverslip, then click Set Offset. The ZDC Single Shot AF window (1) should become activated (2). If it does not work adjust the focus until it does.

    Image Added

  16. Then click “Return to current position” to check the ZDC is working in the Focus Search mode

  17. Make sure “Enable ZDC AF during time series scan” is checked (ZDC does not work without time series)

  18. Press run experiment to start your time series (AF status will show “Searching…” during Focus Search)

  19. When the acquisition has finished click series done

  20. Alternatively you can select “Turn on continuous AF” after setting the Offset and then start your image acquisition (AF status will show “Focussing” throughout the entire image acquisition)