Conceptual
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Spectral Overlap and Unmixing

Broad fluorophore emission spectra bleed into neighboring detection channels, so single-stain reference spectra plus an autofluorescence spectrum are used to linearly unmix a multispectral image into per-marker signals. Without unmixing, a bright marker is misread as co-expression of a dim one.

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Broad fluorophore emission spectra bleed into neighboring detection channels, so single-stain reference spectra plus an autofluorescence spectrum are used to linearly unmix a multispectral image into per-marker signals. Without unmixing, a bright marker is misread as co-expression of a dim one.

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