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Research Technologies Branch

Biological Imaging Section

Protocols: Multiphoton Startup Instructions

The startup and calibration procedure for multiphoton imaging on the Leica SP2 AOBS MP follows:

  1. Check to see that the Mai Tai power supply and water bath are operating normally. These should always be turned on and off together. In any case the water bath should be started before the laser power supply. Under normal conditions, these two units should be left running at all times.
  2. Turn on and boot up the notebook computer that controls the Mai Tai laser. Login is Administrator, password = none. Start Spectra Physics software. The Mai Tai laser is com port 3.
  3. Start confocal system in usual manner and get image in focus using visible laser excitation.
  4. Open confocal pinhole to maximum (4.95).
  5. Use beam expander 6. Switch to beam expander laser 3 if power levels are low.
  6. Switch EOM control box switch "pulse" to low.
  7. Increase gain on PMT 2 or 3 to 850; open detector range to wideband.
  8. Adjust EOM offset to get minimum signal (using QLUT mode).
  9. Switch EOM control box switch "pulse" to pulse.
  10. Adjust gain to minimum power for good signal for use of NDD (non-descanned detectors).
  11. Rotate filter turret to NDD cube position. Turn on NDD detectors in LCS software (multicolored detectors on the left of the instrument control screen).
  12. Turn off epifluorescence lamp power supply.
  13. Adjust gain of each detector to good signal. Note NDD detector 2 is very light sensitive to long wavelengths. It may be necessary to turn off all sources of illumination near the microscope—including monitors!
  14. Adjust the wavelength of the Mai Tai laser to achieve good balance between the fluorochromes. If there has been a large change of wavelength from where the EOM was calibrated, it may be necessary to recalibrate the EOM for the new wavelength.

Note: Please specify which NDD internal filter cube is required. The choices are DAPI/FITC or FITC/TRITC. This must be in place before turning on the system.

  • Based on the wavelengths to be used, you must decide which epifluorescence filter cube is to be replaced by the NDD filter cube. For FITC/TRITC, replace the UV filter cube.
  • It also is possible to use the glycerol immersion 63x lens for multiphoton work. This objective is best matched in RI to large blocks of tissue. This must be installed before setting up the MP system. Mix up glycerol solutions ranging from 50% to 80% for use as an immersion medium.
  • The 20x immersion objective works very well for low magnification multiphoton work. This lens can be used with water, glycerol, or oil as an immersion medium. Please request that this objective be installed if it is required.

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Last Updated April 05, 2007