Method for classification of pixels into groups according to their spectra using a plurality of wide band filters and hardware therefor

A method of classification of pixels into groups of pixels according to their association with a single fluorophore or a combination of fluorophores selected from a plurality of fluorophores, each of the fluorophores having characterizing excitation and emission spectra and specifying excitation and emission peaks, the method comprising the steps of (a) providing a plurality of pairs of wide-band excitation filters (114) and wide-band emission filters (116); (b) exciting fluorophores of each of the pixels with light filtered through one of the wide-band excitation filters, and recording emitted light intensity (122) as retrieved after passing through its paired emission filter; (c) repeating step (b) for all of the plurality of pairs of filters, such that each of the pixels is representable by a vector of a plurality of dimensions, the number of dimensions being equal to the number of the plurality of pairs of filters; (d) using an algorithm for evaluating the presence of each of the plurality of fluorophores in each of the pixels, thereby classifying each of the pixels into a group of pixels according to its association with a single fluorophore or combination of fluorophores.