Time-resolved Fluorescence Spectroscopy System Research Articles

Development of a Steady-State Fluorescence Spectroscopy System and a Time-Resolved Fluorescence Spectroscopy System

This document describes thoroughly the construction of two spectroscopic systems: One system for obtaining fluorescence spectra and another system for obtaining the fluorescence lifetime. The first system consists of obtaining the fluorescence spectrum and is constituted by a Czerny-Turner type monochromator, a photomultiplier tube (PMT), a system for modulating the excitation signal and a system for treating and filtering the fluorescent signal constituted by an electromechanical chopper, a lock-in amplifier and a fast response PMT; plus a set of lenses and a samples holder. The fluorescein dissolved in ethanol was used as a calibration standard, and an Nd:YAG laser source was used as a source of excitation, which emits in 532 nm, of continuous wave and stable power in time adjustable by current. On the other hand, a fluorescence time-resolved system was developed. This system consists of obtaining the fluorescence lifetime of different samples under study from a Voltage curve Vs Time obtained from the system and showing the decay behavior of the phenomenon. This has a pulsed laser unit as a source of excitation, emitting at 532 nm, 80 mJ of energy and with a pulse width of 4 ns. For this system, two semiconductor photodiode detectors with fast response have been built; both with a response of temporary uptake above 1 ns, thus providing obtaining fluorescence lifetime up to this value. Fluorescein was also used in this system as a standard sample, but in this case dissolved in a buffer-like solution; accompanied also by a brief study of the change in the average lifespan of fluorescence in function of the pH of the solution. The results obtained show that two automated systems, reproducible, accurate and of very low cost compared to the current ones, have been successfully implemented for the study of steady-state and time-resolved fluorescence spectroscopy.

Time-resolved fluorescence spectroscopy for the diagnosis of oral lichen planus.

Lichen planus (LP) is a T-cell mediated autoimmune disorder of unknown aetiology that affects the skin, nails, oral and genital mucous membranes. Conventionally, oral LP (OLP) is diagnosed through clinical assessment and histopathological confirmation by oral biopsy. To explore the use of time-resolved fluorescence spectroscopy (TRFS) to detect fluorescence lifetime changes between lesional OLP and perilesional normal mucosa. In this pilot study, measurements of lesional and perilesional buccal and mouth floor mucosa were conducted in vivo with a TRFS system. Histopathological findings were consistent with OLP in 8 out of 10 patients biopsied. Two patients with histopathological diagnoses of frictional hyperkeratosis and oral candidiasis, respectively, were excluded from the study. Our preliminary data show that lifetime values in the 360-560 nm spectral range indicate a significant differentiation between normal and diseased tissue. In contrast to the standard oral biopsy procedure, this technique is noninvasive, painless, time-efficient and safe. Future studies are needed to better elucidate the diagnostic capability of TRFS and to further explore the sources of fluorescence contrast. This pilot study suggests that, based on fluorescence lifetime parameters, TRFS is a very promising technology for the development of a novel OLP diagnostic technique.