Zeiss LSM 880, Operating Manual

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CHAPTER 1 - SYSTEM OPERATION
LSM 880 Center Screen Area / Image Containers - Display and Image Analysis ZEISS
10/2014 V_01 000000-2071-464 499
6.17.1 Automatic Component Extraction
The use of this function permits the automatic
search for the individual reference spectra in a
Lambda stack.
•
Load or acquire a Lambda stack data set.
•
Click on the
Unmixing View
tab.
•
Click on the button in the
Unmixing View Option control tab (Fig. 692).
•
Select the number of components in the sample
and click
Ok
(Fig. 691). If a subset of existing
spectra has been already defined by manual
ROIs or by spectra from the spectra database,
the number of remaining components is
selected for ACE.
•
Review the results in graph.
•
When obtained spectra appear reasonable,
unmix the data set by clicking the
button (Fig. 692).
−
A new and spectrally unmixed multichannel
image document is created (as many
channels as previously defined components).
Benefit and limitations of the ACE method:
Little to moderate spatial overlap of emission signals is required to obtain good results.
Because it relies on reference spectra extracted from the multi-channel / Lambda stack image itself, ACE
could unmix on wrong assumptions and produce poor data.
However, it provides a solution for Emission Fingerprinting in those cases where reference spectra are not
accessible via single-labeled controls.
If the resulting data is carefully checked for plausibility and quantification of the results is avoided carried
out with the appropriate caution, this often is a helpful tool when working with spectrally overlapping
dyes.
Fig. 691 ACE, number of components
selection window
Fig. 692 Automatic Component Extraction
function