Excitationfingerprinting - Zeiss LSM 880 Operating Manual

LSM 880
2.5

ExcitationFingerprinting

With tunable multiphoton lasers (except OPO devices) it is possible to acquire Excitation Spectra and
subsequently use these spectra to perform linear unmixing.
This process is called Excitation Fingerprinting.
To perform this task the software of the system provides the Macro "ExcitationFingerprinting60.lvb"
(available in the folder: C:\ZEN\Macros).
Calibration of the AOM:
A typical Ti:Sa laser does not supply an even power level over the wavelength range. To use such a laser
for Excitation fingerprinting it is recommended to calibrate for the average power at the sample plane
using a power meter.
The resolution of the power meter should be below 1 mW.
For the calibration one channel must be active otherwise the scan will not start and no laser is available.
When ZEN is started one channel is always active per default. If the system was in use, check in the
Imaging Setup tool if a channel is active.
For the calibration it is not relevant which channel is active.
Activate the laser to be calibrated in the Channels tool.
Next, set up the power meter in specimen plane using the combination of objective and main dichroic
that will be used for Excitation Fingerprinting. Both, objective and main dichroic, influence the average
power at the sample as well and need to be taken into consideration for this step.
Set up the Acquisition mode for Frame scanning with maximum speed and bidirectional scanning at the
highest zoom.
This mimics a spot scan and will result in a steady beam coming out of the objective. The read out of the
power meter will be much more accurate.
It is not needed to measure absolute powers but relative powers influenced by the AOM settings.
Load and run the Macro from the Macro Control window.
10/2014 V_01
CHAPTER 2 - MACROS AND VISUAL BASIC
Macros
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ZEISS
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