Precise Analysis of the Autofluorescence Characteristics of Rat Colon Under UVA and Violet Light Excitation

Keimei      Nakano; Yoshinori      Harada; Yoshihisa      Yamaoka; Kiichiro      Miyawaki; Katsuichi      Imaizumi; Hideyuki      Takaoka; Masaya      Nakaoka; Naoki      Wakabayashi; Toshikazu      Yoshikawa; Tetsuro      Takamatsu

doi:10.2174/1389201011314020007

Abstract

Purpose: Tissue autofluorescence study is a promising means of endoscopic detection of colonic neoplasia, but the mechanism of autofluorescence eruption has still not been verified. The purpose of this study was to precisely analyze the autofluorescence characteristics of freshly prepared normal rat colon under UVA and violet light excitation. Methods: Excised rat colons were studied by using multichannel spectrophotometry, spectroscopic imaging, confocal microscopy, combined two-photon excited fluorescence and second-harmonic generation (SHG) microscopy, and fluorescence lifetime imaging microscopy. Results: Spectroscopic analysis of freshly prepared colon sections revealed that the mucosa and the submucosa showed strong autofluorescence under UVA and violet light excitation. The combined images of two-photon and SHG microscopy revealed that the mucosal epithelia are the important source of autofluorescence. Nicotinamide adenine dinucleotide seems to be one of the major substances involved in the autofluorescence of the mucosal layer on 365- nm light excitation. The autofluorescence spectra of the luminal surfaces were identical to those of the mucosa on crosssectional examinations with 365-nm excitation. The main origin of autofluorescence of the luminal surface with 365-nm excitation is the epithelial cells in the mucosa without overlay of submucosal fluorescence. Conclusions: The mucosal layer is the important source of the autofluorescence observed under excitation with UVA/violet light in multilayered colonic structures. Illumination of 365-nm wavelength light is a suitable means of analyzing the autofluorescence of mucosal epithelia.

Keywords: Fluorescence lifetime imaging, mucosal layer, second-harmonic generation, nicotinamide adenine dinucleotide

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