Human tissues intrinsically contains many fluorophores, as such NADH, elastin, collagen, and flavins, that can be
excited and imaged using multiphoton microscopy, up to 150 microns depth. In this work we used combined two photon
intrinsic fluorescence (TPE), second harmonic generation microscopy (SHG), 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 spectroscopic analyses allowed to characterize both healthy and pathological
tissue samples in a good agreement with common routine histology. In particular, we examined tissue samples from
bladder normal 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 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.