MULTIPHOTON LASER SCANNING MICROSCOPY
LSM 510 META NLO
Troubleshooting Checklist
Carl Zeiss
03/06
B 45-0021 e
9-25
9.4 Troubleshooting
Checklist
Several 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 produced
Laser mode-lock:
Check to see if the laser is turned on and that it is mode-locked. For Coherent Chamelon and Spectra
Physics 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 the
percent transmission slider. If no image is seen and the laser is on, check the gain on the PMT and then
increase the laser power. Check to see if increasing the percent transmission increases the power to the
sample (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 card
on 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 may
be closed. Check to see if there is a normal level of power going into the scan head. If no beam can be
found entering the scan head, the shutter or the AOM may not be working and a service engineer should
be 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 or
expert user. Partial illumination of the card reflects poor alignment and the beam should be realigned
into the scan head.
Non-linear power attenuation:
If changing the percent transmission leads to non-linear power attenuation, the AOM can be linearized
using the AOTF fit macro. By opening this macro and following the instructions, one can verify the
linearization of any of the laser lines and then run a linearization protocol to reoptimize the settings. Be
sure the laser is set to the appropriate wavelength before running this macro. Choose only the NIR
wavelength to optimize the AOM attenuation for the Ti: Sapphire laser.
