A computer-implemented multispectral imaging method for use in analysis of a sample comprising a plurality of types of fluorescent
label, each of the plurality of types of fluorescent
label having a respective
emission spectrum is disclosed. 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 the sample and having a respective spectral content; for each channel: i) forming a vector of measured
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 for the contribution made by each of the plurality of types of fluorescent
label to the unfiltered image, and for each iteration, calculating a vector of expected
quantum particle counts, having an entry for each pixel in the image, by multiplying the vector of possible values for each pixel by a mixing matrix defining the relationship between the unfiltered image and the multi-channel image data; and iii) selecting the vectors of possible values for which a negative log-likelihood function describing the probability of a vector of measured
quantum particle counts being generated given a corresponding vector of expected
quantum particle counts is a minimum; and for each of the plurality of types of fluorescent label in the sample, constructing a corresponding
data structure comprising image data in which, for each pixel, the
data structure includes the entry for the contribution made by the type of fluorescent label from the vector of possible values for the pixel, each
data structure thereby being useable to reconstruct an image of the sample with a spectral content corresponding to the respective
emission spectrum of the type of fluorescent label for which the data structure was constructed.