Biology & Biochemistry Imaging Core (BBIC)

Leica TCS SP8 Startup / Shutdown

Turning on the System

  1. Uncover the microscope, make sure that the 10x objective is in the home position, and that the stage insert is OFF if the scanning stage is installed.

  2. Turn on the mercury lamp power source (#1): The green power light and the yellow shutter light will come on. The first and second intensity levels are usually sufficient to observe fluorescence through the eyepieces. Once turned on, the lamp should remain on for a minimum of 30 minutes. Do not turn the lamp off if another user is scheduled within 2 hours after your use.

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  1. Switch on the First green button (#2) (PC/Microscope) on the TCS control panel. The computer will start automatically.

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  1. After 15 seconds, switch on the buttons/key in the following order:
    1. Scanner Power (#3)
    2. Laser Power (#4)
    3. Turn the laser key clockwise from OFF to ON (Laser Emission). (#4)
  2. Switch on microscope control panel (small silver switch on the back). The CRT6500 box should be on (we leave it on), but check it.

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  1. Log into Windows
  2. Start LAS AF software

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  1. Work mode selections:
    1. For normal confocal work: If you want to tile scan (and the scanning stage is installed), select “machine with x-y stage”. Otherwise select “machine no x-y stage”. (if doing Ca imaging you will select “Hamamatsu”). If the translational stage is on, you need to select “machine no x-y stage”.
    2. Microscope: choose DMI 6000 (used normally)
    3. Resonant: for the option of using the resonant scanner. Normally this will be off for fixed samples and on for “fast” live-cell imaging.
    4. Choose your settings, then click OK.

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  1. Microscope stand: This window will appear ONLY if you have selected “machine with x-y stage”. If you are doing a tile scan, click yes, and watch the machine carefully (so that the stage does not hit any objectives). You can click “no” if you selected “machine with x-y stage” instead of “machine no x-y stage” by mistake. This option will be disabled if the translational stage is installed.

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  1. Laser choice: In configuration tab (please leave off lasers that you will not use).

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  1. Turn lasers on in the acquisition panel. Use slider bars to select the laser power for the desired wavelengths.

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End Your session

Before you turn off the microscope, check if there are other people will use the microscope after you. If you cancel your reservation, please do it 3 hours before your reservation time, otherwise please contact the people before you or the Core manager to insure that they don’t leave the microscope on for you.

But whether you are just closing the software, or shutting down the whole system, the first step is always CLEAR THE STAGE. Remove your slides/ specimen holders; take out the stage insert if you are using the scanning. stage.

  1. If there are scheduled users in the next 3 hours:
    1. Turn lasers off in acquisition panel. Set slider bars to 0%.
    2. Open the “Configuration” tab and set the Argon power slider to 0%.
    3. Click FILE/EXIT to terminate the LAS software. Close all open windows and exit the user profile you are using but do not turn the computer off.
    4. Fill out the log book.
    5. Leave the PC, scanner and lasers on. Clean up the objectives; have the lights turned on so that you can see properly. Use lens paper but do not rub.
  2. If there are no scheduled users in the next 3 hours, you should completely turn off the system:
    1. Set all lasers to 0% and turn off in acquisition window
    2. Open the “Configuration” tab, set argon laser power to minimum and then set all lasers to the off position.
    3. Exit LAS software and turn off the computer from start menu
    4. Turn off microscope control panel (#6)
    5. Turn off fluorescent light source (#1)
    6. Turn off scanner and PC power. Turn the “Laser emission” key (#5) counter-clockwise to off, but leave the laser power button (#4) on for at least 5 minutes. This keeps the fan running which helps cool the Ar laser and will maximize its useful life.
    7. Fill out the log book.
    8. Clean objectives with lens paper provided by the core (and nothing else!), wipe objectives in a circular motion, but do not rub! Use a separate square of lens paper for each objective you clean. Put the 10x objective in the home position.
    9. Cover microscope with the dust cover
    10. Wait at least 5 minutes for the fan to slow down, then turn off laser power (button #4)

Always clean up whatever mess or spills may have occurred during your session.

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