Two-photon microscopy has been successfully used to image several types of tissues, including skin, muscles, tendons.
Nevertheless, its usefulness in imaging bladder tissue has not been investigated yet. In this work we used combined twophoton
excited fluorescence, second-harmonic generation microscopy, fluorescence lifetime imaging microscopy, and
multispectral two-photon emission detection to investigate different kinds of human ex-vivo fresh biopsies of bladder.
Morphological and spectroscopic analyses allowed to characterize both healthy mucosa and carcinoma in-situ samples in
a good agreement with common routine histology. Cancer cells showed different morphology with respect to the
corresponding healthy cells: they appeared more elongated and with a larger nucleus to cytoplasm ratio. From the
spectroscopic point of view, differences between the two tissue types in both spectral emission and fluorescence lifetime
distribution were found. Even if further analysis, as well as a more significant statistics on a larger number of samples
would be helpful to discriminate between low, mild, and high grade cancer, our method is a promising tool to be used as
diagnostic confirmation of histological results, as well to be implemented in a multi-photon endoscope or in a
spectroscopic for in in-vivo imaging applications.