The present invention relates to a method of optically imaging at least one biological tissue, in particular to define areas of interest of tissue(s) to be analyzed by tomography.The method according to the invention comprises the following steps: a) introducing at least one fluorescent marker into the tissue(s); b) exciting the marker by incident light and detecting emission bands relating to fluorescence emitted by the marker in response to that excitation; then c) analyzing the fluorescence in these emission bands; and the step b) comprising: sequentially exciting said marker at n different incident excitation wavelengths λi, said marker being adapted to be excited by at least two of the wavelengths λi and to emit in response to each wavelength λi a series Si of m simultaneous emission bands Bj having different maximum wavelengths λ′j that are substantially the same from one series Si to another; and detecting these series Si in order to deduce therefrom an estimate of the three-dimensional location of said marker in the tissue(s) and/or the mean absorption coefficients of the tissue(s) for the excitation wavelengths λi.