Method of fluorescence imaging including: illuminating a sample to excite its fluorescence and acquiring an image thereof; based on fluorescence spectral and spatial information from the sample's fluorescence image, segmenting the image into regions of similar spectral properties; for each image segment, exciting the fluorescence of the corresponding sample region, and detecting the corresponding fluorescence; based on modelling, determining expected fluorescence parameters from the fluorescence signals detected for each region; scanning the sample and determining final fluorescence parameters based on said expected fluorescence parameters.