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Removing the “Haze” Signal
In fluorescence images, image contrast and unmixing
accuracy can sometimes be improved if the general haze
in the image, which has a characteristic spectra, is
subtracted from the images first. When creating the
spectral library for the images, create the background or
“haze” spectrum and add it to the library. When the library
is loaded, you can select the haze spectrum and remove it
from the images.
To remove haze:
1. Check the Remove Haze check box.
2. Select the Haze Spectrum to remove.
Haze is removed numerically (without scaling or other
changes in value), wavelength-by-wavelength, from every
pixel of the image. This alters the spectral shape of the
remaining signals and their overall intensity. The
magnitude of these changes depends on the intensity of
the spectral signal being subtracted. Typically, when
background spectra are small compared to the signals,
spectral library entries derived from unsubtracted data sets
can still be used, but this should be validated when
quantitative results are required. Ideally, the remaining
spectra in the library should also have the haze spectrum
subtracted (in Nuance).
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Figure 14. Remove Haze
Spectrum