MULTIPHOTON LASER SCANNING MICROSCOPY
Carl Zeiss
Using the LSM 510 NLO direct coupled system
LSM 510 META NLO
9-14
B 45-0021 e
03/06
9.3.2
Alignment of the ultrafast laser into the LSM 510 NLO scan head
The alignment of the laser is done by a Zeiss service engineer. However it might be needed to slightly
adjust the beam coupling from time to time, depending on the room conditions. The protocols
mentioned should only be performed if the user is confident with the technique, otherwise the coupling
of the laser can be lost and a Zeiss service engineer is needed for realignment.
The alignment of the laser through the AOM and into the scan head is critical for producing high quality
images using multiphoton excitation. Below are two protocols that can be used to optimize alignment of
the beam into the scan head.
9.3.2.1
Quick alignment protocol
After the system is installed and aligned by a qualified service engineer, it is often the case that only small
alignment adjustments are necessary. For instance, proper alignment should be verified after the laser is
tuned to a new wavelength. Below is a quick and easy protocol that can be used to center the beam into
the back aperture of the objective lens for optimum image quality.
Start by scanning a sample that is normally excited by the wavelength of the external laser. For instance,
a pollen grain slide (Carolina Biological) or a Fluocells sample (Molecular Probes) can be used. (NOTE: This
procedure may cause bleaching of the sample.)
Adjust the laser power and gain so that the sample is visible in the image acquisition window.
Scan the sample using the fast XY scanning mode.
Using the signal intensity as a guide, peak the alignment of the beam. Begin by using the alignment
mirror closest to the laser (first turning mirror). Make improvements using both alignment pins on the
mirror mount. (NOTE: a small hex wrench (Allen key) may be needed to adjust alignment). When the best
signal intensity is obtained by tweaking the mirror closest to the laser, now make improvements by
tweaking the mirror closest to the microscope scan head. Go back and forth between these mirrors to
walk the center the beam onto the back aperture of the lens.
As a final verification of proper alignment, check the alignment of the beam onto the back aperture
directly by viewing the projected beam without the objective in place. To do this, place a white card on
the stage (Fig. 9-5/
A
) or lens paper on the specimen holder for an inverted microscope.
Remove one objective, select 50-100 % transmission, HFT KP 680 or 700, and Fast XY scan. When the
system is scanning, a round, red spot should appear on the card (Fig. 9-5/
B
). Seeing this beam may be
aided by using an IR viewer, especially at longer wavelengths.
