Setup Of Tracks Using Single Detectors (Channel Mode) - Zeiss LSM 880 Operating Manual

ZEISS
Fig. 1 Imaging Setup tool
• Assign appropriate colors to these activated channels.
Example: Ch1 - green (for Alexa 488FITC emission, Ch2 (or ChS1) - red (for CY3 Alexa Fluor 568
emission)
b) Multiple tracks – sequential acquisition of channels
Multi-tracking is the method of choice for multi-fluorescence imaging. It allows avoiding artifacts from
emission crosstalk that can occur in simultaneous excitation and detection of multiple dyes. To
sequentially acquire multiple channels, laser lines are switched very fast and channels are recorded quasi-
simultaneously.
The configuration of multiple tracks follows the same rules described above for a single track. The main
difference is that each track is configured to excite and detected only one fluorescent dye to prevent
cross talk (or two dyes with non-overlapping emission spectra).
• Create a track for Alexa FITC.
6
CHAPTER 4 - ANNEX
Beam path Configuration Guide for ...
3.2
a) One track only – simultaneous acquisition
of channels
• Switch on the suitable lasers for excitation of
• Activate the appropriate laser lines in the Laser
• Select a main dichroic beamsplitter (MBS) which
• Select the proper emission bands in front of the
The active detection bands may not include any of
the active laser lines however this will not interfere
with imaging.
Additional hints:
• Do not forget to turn on the corresponding
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Setup of Tracks Using Single
Detectors (Channel Mode)
the dyes in the specimen in the Laser tool.
Example: for FITC and AlexaFluor 568 use 488
and 561 nm for excitation.
panel by checking the box of the laser line and
set the transmission for each active line using
the slider next to
wavelength.
Example: Select 488 to 0.5% and 561 to 2 %
deflects the selected laser lines to the specimen.
Example: MBS 488/561
channels and activate channels.
Example: select 589 - 554 nm in front of Ch1
and 561 - 669 nm in front of ChS1 (or Ch2).
lasers and detectors.
LSM 880
the indicated laser
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