Processing - Linear Unmixing - Zeiss LSM 880 Operating Manual

ZEISS
5.4.8 Processing – Linear Unmixing
 standard
Tool accepts lambda stacks and multi-channel image data (single images and multi-
dimensional image data). Linear Unmixing was developed for processing LSM data.
Successful processing of multi-channel images obtained with other systems is
potentially possible as well (provided this input meets the requirements for unmixing).
See full description of tool for details.
Fig. 409 Processing – Linear unmixing
Finally, the individual components are mathematically extracted using the information from the reference
spectra. Up to ten different reference signals can be fed into the least-square-fit based algorithm to
produce a 10-channel multi-fluorescence stack without any partial overlap between the channels.
If no mono-labeled samples are available, the references can be obtained by the following methods:
a) Interactively by user-selection of regions in the image where only one fluorescence dye is present
(only available in the Unmixing View tab).
b) Automatically by software analyses of what the individual spectral signatures are. This processing
function is called "Multi-channel Unmixing" or "Automatic Component Extraction" (ACE). Note: in
some cases, spectrally acquired images are not appropriate for ACE or "Multi-channel Unmixing"
and linear unmixing can lead to wrong results.
• To open the Linear unmixing tool, click on Linear unmixing in the Processing tool list under the
Processing tab (Fig. 409).
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CHAPTER 1 - SYSTEM OPERATION
Left Tool Area and Hardware Control Tools
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Instrument
 LSM ELYRA
The Linear Unmixing processing tool permits
extracting the emission of single fluorescence dyes
(e.g. GFP only, YFP only etc.) from strongly
overlapping multi-fluorescence data acquired in
multi-channel images and so-called "Lambda
stacks" (only available in LSM imaging mode).
In brief, with the knowledge of the spectral
characteristic of individual components within a
multi-component sample, even heavily overlapping
individual spectral
mathematically extracted from an experimental
multi-channel data. This method is a strictly pixel-
by-pixel image analysis procedure.
Experimentally, fluorescence spectra of mono-
labeled samples are acquired and stored in the
Spectra Database as an external reference. Then a
multi-channel image or Lambda stack (available
only in LSM imaging mode) from the multi-
labeled sample is acquired.
LSM 880
Lightsheet Z.1
characteristics can
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be