Fluorescence tomography is a preclinical and clinical imaging method which aims to localize fluorescent probes injected into a living organism. In this thesis work, we defined the optimal design and parameters of a continuous wave fluorescent tomograph applied to thin tissue observation. We handled the problem of observing heterogeneous and highly absorbing organs. Secondly, we showed the advantage of time of flight measurement for inclusion detection through thicker tissues. The detection performances of two types of system capable of these measurements were compared and we proposed a protocol to optimize their main parameters. This process was applied to the problematic of prostatic biopsy guiding. It would also be used to detect and localize other pathologies by means of a simple adaptation.