Time-resolved spectrofluorometer for clinical tissue characterization during endoscopy

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Abstract
Time-resolved fluorescence spectroscopy has the potential to provide more information for the detection of early cancer than continuous wave spectroscopy. A new optical fiber-based spectrofluorometer for time-resolved fluorescence spectroscopy of biological tissue during clinical endoscopy is presented. The apparatus is based on a nitrogen laser pumping a dye laser as excitation source and a streak camera coupled with a spectrograph as time-resolved spectrometer. The excitation and fluorescence light is carried by an optical fiber to the tissue under investigation and back to the detector, respectively. This optical fiber can be inserted into the biopsy channel of a conventional endoscope. Hence, the apparatus can be used to perform in situ tissue characterization during endoscopy. The instrument enables the measurement of the decays of entire fluorescence spectra within 15 s with a dynamic range of the spectro-temporal images of up to three orders of magnitude. Luminescence lifetimes from the sub ns up to the ms range can be measured. Spectral and temporal resolution, sensitivity, and dynamic range of the instrumentation were determined. The accuracy of the apparatus was checked by the measurement of the fluorescence lifetimes of various fluorophores with known lifetimes. For the first time, two-dimensional time-resolved spectra with sub-ns temporal resolution of tissue fluorescence of the human bladder, the bronchi, and the esophagus taken during endoscopy are presented as a demonstration of performance of the instrumentation. The excitation wavelengths were 337 nm in the case of the bladder and the esophagus and 480 nm in the case of the bronchi. Lifetime contrasts between normal and neoplastic tissue were found in all three organs. The spectral analysis of the fluorescence decays showed that the fluorescence between 370 and 490 nm, excited at 337 nm, consisted in several overlapping spectra. In the case of the esophagus, the contrast between normal and tumoral tissue was inverse in two different spectral bands proving the importance of the choice of the appropriate spectral range for time-resolved autofluorescence measurements for an optimal contrast. The in vivo fluorescence decay of the photosensitizers 5-aminolevulinic acid hexylester hydrochloride-induced protoporphyrin IX was measured in the human bladder and found to be mono-exponential with a lifetime of 15.9 (±1.2) ns. An in vivo fluorescence lifetime of 8.5 (±0.8) ns was found in the case of the photosensitizer 5, 10, 15, 20-tetra(m-hydroxyphenyl)chlorin (mTHPC) in the esophagus.