Time-resolved confocal fluorescence spectroscopy reveals the structure and metabolic state of epithelial tissue

Abstract

Autofluorescence spectroscopy has been a widely explored technique for in vivo and noninvasive diagnosis of pre-cancer lesions in epithelium where 90% cancers originate. For extracting more accurate fluorescence information for cancer diagnosis, depth-resolved fluorescence measurements are crucial to assess NADH and FAD in non-keratinized epithelial layer and collagen in stromal layer, respectively. In this study, we achieved the depth-resolved fluorescence spectral measurements of squamous epithelial tissue based on confocal technique. We found that in non-keratinized epithelial layer the fluorescence signals excited at 405 nm were the combination of NADH and FAD fluorescence and could be used for evaluating the redox ratio. Moreover, we found that confocal time-resolved autofluorescence measurements of epithelial tissue with 405 nm excitations could provide the information on the layered tissue structure. All depth-resolved autofluorescence decays were accurately fitted with a dual-exponential function consisting of a short lifetime (0.4 ~ 0.6 ns) and a long lifetime (3 ~ 4 ns) components. The short lifetime component dominated the decay of non-keratinzied epithelial fluorescence while the decay of the signals from keratinized epithelium and stroma were mainly determined by the long lifetime component. The ratio of the amplitudes of two components could be used to differentiate the layered structure of epithelial tissue. In general, the results in this study demonstrated that the combined depth- and timeresolved fluorescence measurements can produce the information on the layered structure and localized biochemistry of epithelial tissue for the diagnosis of tissue pathology.