Zeiss LSM 880, Operating Manual

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CHAPTER 5 - MULTIPHOTON LASER SCANNING …
LSM 880 NLO (Non Linear Optics) Laser for LSM ZEISS
10/2014 V_01 000000-2071-464 11
1.4 Pinhole and Collimator Settings
•
The pinhole can be fully opened for maximum detection efficiency due to the intrinsic optical
sectioning capabilities of the NLO effect (see
Channels tool
).
•
In the
Adjust Pinhole & Collimator
tool, the
NLO
collimator can be used to align the overlap of the
excitation planes within the object for VIS as well as NLO excitation light.
•
Select the NLO collimator in the Collimator panel. It is best to use a fine structured object like beads
which are excited with visible lasers and the NLO laser.
•
Focus on the bead and acquire an image with the visible laser, for example 488 nm excitation. Keep
the image and open a new image document in a separate image container.
•
Then switch to the NLO laser and start a continuous scan.
•
Activate the Profile View tab.
•
Move the position of the collimator (detaching the tool window so it is visible) such that the intensity
of the bead is at its maximum and matches the image of the bead taken with the visible laser.
•
Now the excitation planes are matched between the visible and the NLO laser.