OPERATION IN EXPERT MODE
LSM 510
DuoScan
Carl Zeiss
Process Menu
LSM 510 META
DuoScan
4-146
B 45-0021 e
03/06
4.6.11 Unmix
The
Unmix
functionality permits to extract the emission of single fluorescence dyes (e.g. GFP only, YFP
only etc.) from the overall emission band of strongly overlapping multifluorescence signal intensities by a
pixelwise linear unmixing procedure .
Mathematically, experimental fluorescence spectra of monolabelled samples are taken as an external
reference. Up to 8 different reference signals can be varied in this least-square-fit based algorithm to
produce an 8 channel multifluorescence stack without any partial overlap between the channels.
4.6.11.1
Open / Close the Unmix Window
•
Click on the
Unmix
button in the
Process
subordinate toolbar of the
Main
menu.
−
This opens the
Linear Unmixing
window.
•
Click on the
Close
button to quit the
Unmix
window.
4.6.11.2 Source
Panel
In the
Source
panel the image source for the linear unmixing process has to be defined.
This has to be a Lambda Stack, a Lambda Stack Z series or a Lambda Stack T series.
Proceed as follows to select an image via the image selection box:
•
Click on the arrow button. The image selection box is opened and all the currently loaded images,
stacks, time series with a Lambda dimension are displayed in a minimized form.
•
Click on the required image. This image will then appear in the display box of the image selection box
and has been selected.
4.6.11.3
Definition of Channels Panel
In the
Channels
panel the number of reference spectra (number of fluorescence channels) can be
selected from the channel selection boxes.
•
Select the references fluorescence dye spectra which are present in the sample with the check boxes.
•
If necessary change the colors of the relevant fluorescence channel.
•
If no predefined reference spectra exist, please define reference signals via the
Save to Spectra DB
button in the
Display
-
Mean
functionality (see page 4-338).
•
After definition of the required reference spectra set click on the
Apply
button.
•
A new window with the resulting channels of the unmixing procedure opens immediately.
There are several settings that can be chosen for linear unmixing.
Autoscale
balances the intensity of the unmixed images.
