Spectral unmixing involves computational techniques to deconvolve overlapping emission spectra. The process typically includes: - Spectral Library Creation: A library of known emission spectra for each fluorophore is created. - Linear Unmixing Algorithm: The algorithm uses the spectral library to fit the observed spectrum at each pixel into a linear combination of the known spectra. - Quantitative Analysis: The contribution of each fluorophore is quantified, allowing for accurate differentiation and analysis.
