MULTIPHOTON LASER SCANNING MICROSCOPY
Carl Zeiss
Troubleshooting Checklist
LSM 510 META NLO
9-26
B 45-0021 e
03/06
9.4.2
Poor image quality
Lines or spaces across the image:
This can result from an unstable mode-lock. CW breakthrough appears as blank lines in the image.
Q-switching will result in wavy lines in the image. A service engineer from Choherent or Spectra need to
check the laser.
Poor fluorescence sensitivity:
This can result from many factors or a combination of factors. These include the following:
Correct wavelength (see 9.3.4):
Be sure that the laser is set at the best wavelength for the fluorochrome being examined. Empirical
testing may be necessary to determine the best wavelength. Tuning to a different wavelength may also
help to reduce autofluorescence from some tissues.
Poor Beam Alignment into the scan head (see 9.3.2.2):
Remove the objective and look at the reflection of the beam onto a white card. The beam should be
round and even without any flat edges. Peak the alignment of the beam into the scan head.
Poor transmission through the objective (see 9.3.3):
Many objectives are corrected for work with UV and visible range excitation and have poor transmission
of photons in the near IR.
Sliders/polarizers in the path:
DIC sliders and polarizers in the path can reduce excitation and emission sensitivity. Remove sliders or
slide them to an open position.
Poor sensitivity in fixed samples:
Fixatives and mounting medias can sometimes limit the sensitivity of fluorescence detection or increase
autofluorescence reducing the signal-to-noise ratio. For multiphoton excitation, the mounting media can
also reduce the peak intensity of the laser deep into the sample. Empirical determination of the best
fixative and mounting media for a particular application may be necessary.
