Zeiss LSM 510 Manual

Also see for LSM 510: Quick start

MULTIPHOTON LASER SCANNING MICROSCOPYLSM 510 META NLO Troubleshooting Checklist Carl Zeiss03/06 B 45-0021 e 9-259.4 Troubleshooting ChecklistSeveral key things can go wrong when imaging that will result in poor or no image being produced.Below is a checklist to help the user produce the best images possible.9.4.1 No image being producedLaser mode-lock:Check to see if the laser is turned on and that it is mode-locked. For Coherent Chamelon and SpectraPhysics Mai Tai lasers this is indicated in the LSM software, Laser control window.AOM not on or too little laser power:Check to see if the laser is turned on in the laser control panel and that the box is checked next to thepercent transmission slider. If no image is seen and the laser is on, check the gain on the PMT and thenincrease the laser power. Check to see if increasing the percent transmission increases the power to thesample (use the monitor diode) in a linear way. If not, the AOM settings may need to be reset using the“linearize AOTF” macro.Inefficient fluorochrome excitation (see 9.3.4):Use a control slide to determine if the problem is in the sample. If excitation is seen with other samples,optimization of the laser wavelength may be necessary.Laser power to the sample. Verify that the laser is focused through the objective by placing a white cardon the microscope stage and removing the objective.A bright red spot approximately 5-7 mm wide should be seen on the white card.If no light is seen, then the beam may not be focused into the scan head or the shutter of the AOM maybe closed. Check to see if there is a normal level of power going into the scan head. If no beam can befound entering the scan head, the shutter or the AOM may not be working and a service engineer shouldbe called. If there is a beam after the 1 m lens, there may be poor alignment going into the scan head.Report this to a service engineer or attempt realignment under the guidance of a service engineer orexpert user. Partial illumination of the card reflects poor alignment and the beam should be realignedinto the scan head.Non-linear power attenuation:If changing the percent transmission leads to non-linear power attenuation, the AOM can be linearizedusing the AOTF fit macro. By opening this macro and following the instructions, one can verify thelinearization of any of the laser lines and then run a linearization protocol to reoptimize the settings. Besure the laser is set to the appropriate wavelength before running this macro. Choose only the NIRwavelength to optimize the AOM attenuation for the Ti: Sapphire laser.