2-photon quick setup / shutdown list

[This document is to serve as a reminder for those who are already trained.]

To start up:

    1. Turn on the 2P microscope power (the big switch).

    2. Turn on the 2P acquisition computer

    3. If you will use epifluorescent imaging, turn on the Xcite illuminator.

    4. On the computer, start the PrairieView application.

    5. On the 2P laser tab (the tabs are about halfway down the PrairieView control panel), click the toggle button to turn the Laser power "ON". Wait for the power to come up to over 1000mW.

To begin 2-photon imaging:

    1. Make sure the turret is in position 1 (the position that is labeled "2P" on the key).

    2. Make sure the selector rod is all the way out (LSM position as opposed to BF)

    3. Make sure the microscope door is lowered as much as is possible for your configuration

    4. Make sure the filter wheel on the Xcite illuminator is set to block the outgoing light

    5. Make sure you have tuned on the PMT channels you want to image

    6. Press LIVE SCAN and make sure you hear the microscope shutter click.

    7. Set the PMT gains for the channels you want to use; a good starting point is 800 units (typical range of use in brain is 700-900)

    8. On the Laser/PMT/DAQ tab set laser power with the "pockels" slider (for pollen, use less than 10mW, usually 3-5 pockels units; for the brain, use <50mW for physiology; see the calibration page to see the relationship between pockels units and laser power (in mW) that is emitted at the objective).

Using the piezo fast Z-scanner:

    1. Leave the piezo powered off while you screw in the objective and mount it on the microscope.

    2. Turn on the power supply box (behind the acquisition computer's monitor) when the piezo is in place.

    3. When finished, turn off the piezo power supply box before removing the piezo Z-scanner.

Clean up:

    1. Be sure the Mai Tai laser emission is turned "OFF" and the Mai Tai shutter is closed (via the "2P laser" panel on the PrairieView software)

    2. Turn off any gases used (including the nitrogen that floats the table)

    3. Clean and store any objectives that were used according to their instructions. The 40XIR or 16X lens should be cleaned by gently touching the business end to lens paper and moving the paper slowly until it is dry.

    4. Make sure the Xcite fluorescent lamp is off

    5. Quit the PrairieView program

    6. Power down the acquisition computer

    7. Power down the 2-photon microscope at the switch on the equipment rack.