Fluorescence spectrum estimation using multiple color images and minimum negativity constraint

An inexpensive method to convert a microscope into an imaging spectrometer is presented. Unlike current microscope-based spectrometers which use specialized optics or scanning mechanisms, our system only requires at most two image captures with a 3-chip CCD camera and a lightly-tinted color filter to output the color signal of a sample at each pixel. Basis spectra are obtained by principal components analysis applied to an ensemble of color signals of commercially-available dyes observed with different dichroic mirrors. A transformation matrix from channel values to spectral coefficients is derived. Minimum negativity constraint is applied to eliminate negative parts of the reconstructed fluorescence spectrum. The technique is demonstrated on fluorescence microspheres (fluorospheres) and chlorophyll from plant leaf.