Zeiss LSM 880, Operating Manual

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CHAPTER 2 - MACROS AND VISUAL BASIC
LSM 880 Macros ZEISS
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The normalized AOM values will provide the right AOM settings starting with the chosen setting at a
given wavelength and calculating the relative values for all other wavelengths. Thus it is avoided to reach
maximum or minimum AOM settings before the end of the wavelength range.
The step size for setting the AOM and tuning the laser can be edited.
Choosing
Manual
requires performing the tuning of the laser and the setting of the AOM by
using the slider or typing in a value.
Single highlighted values in the calibration list or the whole list can be deleted.
Mean intensity (%) refers to the overall image intensity value; the value is displayed as % of the
maximum intensity value at chosen data depth (8, 12 or 16 bit).
The lower part of the window shows the values of the currently used calibration file, the laser status, the
objective and main dichroic.
The
Store wavelength button will update the laser wavelength in the main SW.
Acquisition of an Excitation Lambda Stack:
1) Focus the specimen and set the AOM and detector gain to obtain a good image at the chosen
wavelength. Switch to Excitation Lambda Stack.
2) Load the calibration file required for the used main dichroic and objective.
3) Set the wavelength range and step size at which the images should be acquired. (This is
independent of the range and step size of the calibration curve.)
4) Start the image acquisition.
The Excitation lambda stack is displayed the same way as an emission lambda stack.
When checking the
Show Text option the wavelength used for excitation of each image is displayed.
The Excitation Fingerprinting or the unmixing of the Excitation lambda stack can be performed in
different ways.
It is possible to use already existing Excitation Spectra from previous experiments.
One can also use ACE (Automatic Component Extraction) to define the spectra from the newly acquired
stack and use those for unmixing.
The spectra can also be defined manually by using the drawing tools.
The method to work best and is convenient to use depends on the labelling of the specimen.
If the different fluorescent dyes show no or only moderate spatial overlap in the specimen, ACE works
well.
If the fluorescent dyes are strongly co-localized, it is recommended to use predefined Excitation Spectra
of the individual fluorescent dyes.