Multispectral multiphoton lifetime analysis of human bladder tissue

Human tissues intrinsically contain many fluorophores, as such NADH, elastin, collagen, and flavins, that can be excited and imaged using multiphoton microscopy, up to 150 μm depth. In this work we have used combined two-photon excited fluorescence (TPE), fluorescence lifetime imaging microscopy (FLIM), and multispectral two photon emission detection (MTPE) to investigate different kinds of human ex-vivo fresh biopsies of bladder. Morphological and spectroscopy analyses have allowed to characterize both healthy and pathological tissue samples. In particular, we have examined tissue samples from healthy bladder mucosa, and bladder carcinoma in-situ (CIS), finding both morphological and spectroscopic differences. From the morphological point of view, cancer cells appeared more elongated with respect to corresponding normal cells; they also exhibited a different nucleus to cytoplasm ratio. From the spectroscopic point of view, we have found differences between the two tissue types in both spectral emission and fluorescence lifetime distribution. Even if further analysis, as well as a more significant statistics on a large number of samples would be helpful to discriminate between low and high grade cancer, our method is a promising tool to be used as diagnostic confirmation of histological results, as well as a diagnostic tool in a multiphoton endoscope or cystoscope to be used in in-vivo imaging applications.