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Two-photon fluorescence (TPEF) microscopy is a powerful tool to image human tissues up to 200 microns depth without
any exogenously added probe. TPEF can take advantage of the autofluorescence of molecules intrinsically contained in a
biological tissue, as such NADH, elastin, collagen, and flavins. Two-photon microscopy has been already successfully
used to image several types of tissues, including skin, muscles, tendons, bladder. Nevertheless, its usefulness in imaging
colon tissue has not been deeply investigated yet. In this work we have used combined two-photon excited fluorescence
(TPEF), 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
colon. Morphological and spectroscopic analyses allowed to characterize both healthy mucosa, polyp, and colon samples
in a good agreement with common routine histology. Even if further analysis, as well as a more significant statistics on a
large 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 as a diagnostic tool in a multiphoton
endoscope or colonoscope to be used in in-vivo imaging applications.
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