A computer-implemented multispectral imaging method is disclosed for analyzing a sample comprising a plurality of types of fluorescent labels, each of the plurality of types of fluorescent labels having a respective
emission spectrum. The method comprises: receiving multi-channel image data, each channel in the multi-channel image data comprising image data derived from an unfiltered image of a sample and having a respective spectral content; for each channel: i) forming a vector for measuring
quantum particle counts from the image data for the channel, the vector having an entry for each pixel in the image; ii) iteratively generating, for each pixel in the image, a vector of possible values having entries contributing to the unfiltered image by each of the plurality of types of fluorescent labels, and for each iteration, generating a vector of possible values for each pixel by multiplying the vector of possible values for each pixel by a
hybrid matrix, the
hybrid matrix defines a relationship between the unfiltered image and the multi-channel image data, calculates a vector of expected
quantum particle counts, the vector having entries for each pixel in the image; and iii) selecting a vector of possible values for which, given a corresponding vector of expected
quantum particle counts, a negative log-likelihood function describing the probability of the generated vector of measuring
quantum particle counts is minimum; and for each of a plurality of types of fluorescent labels in the sample, constructing a corresponding
data structure comprising image data, where, for each pixel, the
data structure comprises an entry contributed by the fluorescent
label type for a vector according to possible values for the pixel, such that each
data structure is available to reconstruct an image of the sample, the spectral content thereof corresponds to a corresponding
emission spectrum of the fluorescent
label type for which the data structure is constructed.