6533b7dcfe1ef96bd12732e2

RESEARCH PRODUCT

Simultaneous detection of tissue autofluorescence decay distribution and time-gated photo-bleaching rates

subject

description

ABSTRACT Experimental methodology for parallel measurements of in-vivo skin autofluorescence (AF) lifetimes and photo-bleaching dynamic has been developed and tested. The AF lifetime decay distributio ns were periodically collected from fixed tissue area with subsequent detection of the fluorescence intensity decrease dynamic at different time gates after the pulse excitation. Temporal distributions of human in-vivo skin AF lifetimes and bleaching kinetics were collected and analyzed by means of commercial time-corr elated single photon counting system. Keywords: TCSPC, skin autofluoresc ence, and photo-bleaching. 1. INTRODUCTION Laser induced time-resolved autofluorescence (AF) spectroscopy represents a promising adjunctive technique for in-vivo tissue diagnostics. Biological and biomedi cal applications of the autofluorescen ce spectroscopy have been successfully applied in cell biology and clinical diagnostics for detection of abnormal tissues [1-5]. The most important endogenous s uorophores are molecules widely distributed in cells and tissues, like proteins containing aromatic amino-acids, NAD(P)H, s avins and lipo-pigments [6,7]. The radiative lifetime of each fluorophore is unique, so fluorescence lifetime measurements can provide specific information on fluorophore content and distribution in the tissue. However, in tissue experiments there are a number of de-excitation processes that decrease the characteristic lifetime. Also, tissue fluorescence lifetime value depends on environmental factors e.g. experimental setup, fluorophore localization, skin pH balance, viscosity, temperature etc. [7,8]. Taking into account the aforementione d circumstances, the selective analysis and separation of individual fl uorophores underlying multi-exponential d ecays of in-vivo skin autofluorescence still is challenging problem. Moreover, tissue autoflurescence usually shows the phot o-bleaching effect which is caused by chemical modi Þ cation of the s uorophore due to repeated excitation/emission states. Temporal behavior of skin autofluorescence photo-bleaching can be well described by double exponential function [9]. The mechanism of photo-bleaching effect has not been explained in detail so far; however many studies have focused on purposeful use of photo-bleaching in biomedical applications. Some of the authors successfully applied blea ching effect in Raman spectroscopy for improving the Raman spectra quality by decreasing of tissue fluorescence backgr ound [10,11]. In photodynamic therapy, photo-bleaching of exogenous fluorophores was used as tool for tracking the concentration changes of light sensitizing agents and for individualization of irradiation time during the therapy [12]. In fluorescence microscopy the photo-bleaching widely used to investigate diffusional mobility of fluorescing proteins in living cells [13]. Also some of authors propose the use photo-bleaching as a tool to measure the local strain field in fibrou s membranes of connective tissues [14]. In spite the fact that the mechanism of photo-bleaching is still unclear one can assume that tissue fluorophore might have its own specific bleaching kinetic, which can provide information on fluorophore concentration and environmental factors. Thus, the photo-bleachin g at different time gates after the pulse ex citation might provide additional information for selective analysis and separation of individual tissue fluorophores in combination with fluorescence lifetime spectroscopy. The correlations between tissue AF lifetimes and photo-bleachin g rates are studied in this work. The measurements were collected from different parts of health y skin, nail and benign nevus. Autofluoresc ence decay temporal distributions were approximated by three exponential decay functions, and AF intensity decrease at different time gates after the pulse excitation during the repeated excitation were recorded and displayed on a plot.