Zeiss LSM 880 Operating Manual page 634

ZEISS
The following procedures describe how to adjust positioning and illumination for the acquired images
using the ZEN software.
1.3.1.1 Correction of the illumination profile
The illumination profile indicates whether there is an angular offset of the beam when scanning the
sample. A representation of the illumination profile is depicted when using the profile view tab.
The profile line drawn across the image shows the distribution of the image intensity over the field of
view. The image intensity should be centred in both the horizontal and vertical dimension, respectively.
The illumination can be centred using the output mirrors only.
• The vertical position of the intensity profile plateau is adjusted by clicking on the buttons representing
Axis 1.
• The horizontal position of the intensity profile plateau is adjusted by clicking on the buttons
representing Axis 2.
1.3.1.2 Correction of image position
If a sample is excited using two NLO lasers or an NLO laser and OPO, the corresponding two images may
have a slight offset with respect to each other. This indicates a lateral shift of one of the beams. The
easiest method to correct for this shift is to align one of the beams while the other one is kept fixed. This
is done under observation of the images acquired in Continuous or Live scanning mode.
1. Use a sample with small distinct objects like beads, that can be excited with both wavelengths
applied.
2. Set up a frame wise multitrack where the first track produces the image with one beam, and the
second track produces the image with the other beam.
3. Use the same detector without an emission filter in place as to collect all the fluorescence of the
sample.
4. Use different colours for the images of the two tracks to be able to distinguish the two images.
For example if the NLO laser / Beam 1 shall be aligned with the OPO beam/ Beam 2, then Beam 1 can be
adjusted according to the following procedure (see next pages):
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CHAPTER 5 - MULTIPHOTON LASER SCANNING ...
NLO (Non Linear Optics) Laser for LSM
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LSM 880
10/2014 V_01