Zeiss LSM 880, Operating Manual

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CHAPTER 5 - MULTIPHOTON LASER SCANNING …
ZEISS Non Descanned Detection (NDD) LSM 880
12 000000-2071-464 10/2014 V_01
2 Non Descanned Detection
(NDD)
The use of Non-Descanned Detectors with the
LSM 880 NLO is only useful in combination with
the NLO laser.
The Non-Descanned Detection modules can be
used on the reflected or transmitted-light beam
path (transmission NDDs not available for Axio
Imager) or simultaneously on both beam paths.
This means that up to 10 NDD channels
(depending on the microscope used) can be
configured. If NDD channels have been assigned to
the transmitted-light beam path on Axio Observer,
no transmission PMT can be implemented.
In Non-Descanned Detection, the radiation emitted
by the specimen is directly guided onto the
relevant detector without passing the scanner
mirror again.
Non-Descanned Detection is set and configured in
the
Imaging Setup tool by choosing the Non-
Descanned
tab next to the
LSM
tab. This tab is
only available when NDD detectors are connected.
•
Click on the
Non-Descanned
tab to change to
Non-Descanned Detection.
•
After checking a box next to a channel the
corresponding detection window is indicated in
the bar in the upper part of the window. Within
each bar an arrow opens a drop down window
which allows choosing the fluorescent dye to be
imaged. Its emission profile is then indicated in
the spectral display.
2.1 Thickness of optical slices using multiphoton excitation and non descanned
detection
In the Channels tool the thickness of the optical slices is indicated dependent on the excitation
wavelength of the NLO laser. This slice thickness (FWHM) is calculated according the following formula:
2 𝑥√𝑙𝑙 2 𝑥
0.532
λ
√
2
𝑥 �
1
𝑙 − √𝑙 2 − 𝑁𝐴 2
�
It does not take into account system specific influences on the excitation PSF. This formula is not applied
whenever an internal detector with a pinhole is used even if the pinhole is completely open.
Fig. 5 Light Path tool with NDD detectors
(2 in reflection, 2 in transmission)
on Axio Examiner