Methods and systems are disclosed for extracting an image of a target fluorophore in a biological material, which involve inducing both autofluorescence of the biological material and fluorescence of the fluorophore, acquiring an image arising from both the autofluorescence of the biological material and the fluorophore, and an image arising only from the autofluorescence, subtracting the two images to produce an image representing only the fluorophore, wherein relative intensities of the excitation light used to induce the autofluorescence and the fluorescence are modulated prior to acquiring the images.
