Zeiss Lightsheet Z.1, Operating Manual

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CHAPTER 4 - SYSTEM OPERATION
Carl Zeiss Left Tool Area and Hardware Control Tools Lightsheet Z.1
96 000000-1790-528 02/2013
•
Select the sample-specific fluorescence dye reference
spectra from the Spectra Database (Fig. 142).
•
Assign appropriate colors to the fluorescence
channels by clicking on the color flag and choose
from the available lookup tables (LUTs)
•
If no predefined reference spectra exist:
−
Prepare reference samples and create spectra via
the
Save to Spectra DB button in the Unmixing
View (see section
3.3.7 Processing – Linear
Unmixing ).
−
or
−
Use the "Multi-channel Unmixing" functionality
described below. (Note: in some cases, spectrally
acquired images are not appropriate for "Multi-
channel Unmixing" and linear unmixing can lead
to wrong results – see discussion below.)
•
After definition of the required reference spectra click
Apply
.
A new image with the resulting (unmixed) channels will
be created (Fig. 143 and Fig. 144).
The following additional settings and functions are
available for linear unmixing:
Auto scale balances the intensity of the unmixed
channels to equal levels.
Display Channel with Residuals generates an additional
channel in which the intensity values represent the
difference between the acquired spectral data and the
fitted linear combination of the reference spectra. In
essence, the residual value is the biggest remaining
"residual" from the least square fit routine. The residuals
are a general measure for how good the fit of the
algorithm has performed.
The higher the intensity in this additional channel, the worse is the fit of the spectra to the dataset. This
shows that either the acquired data is not representing the sample (e.g., when information is lost by pixel
saturation) or wrong/insufficient reference spectra have been chosen. In this case, new image acquisition
with no overexposed/ saturated pixels is strongly recommended. In the case of wrong reference spectra,
choosing/acquisition of different/new reference spectra can be necessary to improve the results.
Background: Select a background spectrum from the list of selected components. This spectrum is then
subtracted from the images prior to unmixing. For example: three components selected and one of them
marked as background will subtract the selected background from the acquired images and generate a
2-channel unmixed image. For the generation of background spectra refer to the Unmixing View. Even
small background contributions to the image should be indicated for the unmixing algorithm to ensure
best unmixing results.
Fig. 142 Spectra and LUT assignment