Optical fiber-based setup for in vivo measurement of the delayed fluorescence lifetime of oxygen sensors.

A new optical-fiber-based spectrofluorometer for in vivo or in vitro detection of delayed fluorescence is presented and characterized. This compact setup is designed so that it can be readily adapted for future clinical use. Optical excitation is done with a nitrogen laser-pumped, tunable dye laser, emitting in the UV-vis part of the spectrum. Excitation and luminescence signals are carried to and from the biological tissues under investigation, located out of the setup enclosure, by a single optical fiber. These measurements, as well as measurements performed without a fiber on in vitro samples in a thermostable quartz cell, in a controlled-atmosphere enclosure, are possible due to the efficient collection of the laser-induced luminescence light which is collected and focused on the detector with a high aperture parabolic mirror. The detection is based on a gated photomultiplier which allows for time-resolved measurements of the delayed fluorescence intensity. Thus, relevant luminescence lifetimes, typically in the sub-microsecond-to-millisecond range, can be measured with near total rejection of the sample's prompt fluorescence. The instrument spectral and temporal resolution, as well as its sensitivity, is characterized and measurement examples are presented. The primary application foreseen for this setup is the monitoring and adjustment of the light dose delivered during photodynamic therapy.

Assessment of a sheep animal model to optimise photodynamic therapy in the oesophagus.

BACKGROUND AND OBJECTIVES: Precursor lesions of oesophagus adenocarcinoma constitute a clinical dilemma. Photodynamic therapy (PDT) is an effective treatment for this indication, but it is difficult to optimise without an appropriate animal model. For this reason, we assessed the sheep model for PDT in the oesophagus with the photosensitiser meta-(tetra-hydroxyphenyl) chlorin (mTHPC). MATERIALS AND METHODS: Twelve sheep underwent intravenous mTHPC injection, blood sampling and fluorescence measurements. mTHPC's pharmacokinetics was measured in vivo and in plasma by fluorescence spectroscopy. Biopsies of sheep oesophagus were compared to corresponding human tissue, and the mTHPC's biodistribution was studied under fluorescence microscopy. Finally, the sheep oesophageal mucosa was irradiated, 4 days after mTHPC's injection. RESULTS: Histologically, the sheep and human oesophagus were closely comparable, with the exception of additional fatty tissue in the sheep oesophagus. mTHPC's pharmacokinetics in sheep and human plasmas were similar, with a maximum of concentration in the sheep 10 hours after i.v. injection. mTHPC's pharmacokinetics in vivo reached its maximum after 30-50 hours, then decreased to background levels, as in humans under similar conditions. Two days after injection, mTHPC was mainly distributed in the lamina propria, followed by a penetration into the epithelium. The sheep and human tissue sensitivity to mTHPC PDT was similar. CONCLUSION: In conclusion, this model showed many similarities with humans as to mTHPC's plasma and tissue pharmacokinetics, and for tissue PDT response, making it suitable to optimise oesophagus PDT.

Light-induced retinal vascular damage by Pd-porphyrin luminescent oxygen probes.

PURPOSE: The phosphorescence lifetime of certain metalloporphyrins dissolved in a physiological medium provides an optical signature for local oxygen concentration (pO(2)). This effect is used for measuring physiological pO(2) levels in various tissues. However, the phosphorescence quenching of certain metalloporphyrin triplet states by oxygen also creates singlet oxygen, which is highly reactive and capable of inducing tissue damage. In the current study, the Pd-meso-tetra(4-carboxyphenyl) porphyrin dye (PdTCPP) was simultaneously used as an oxygen sensor and a photosensitizer. Phototoxicity was assessed in the eye fundus and correlated with tissue oxygenation, drug-light dose, and severity of tissue damage. METHODS: The kinetics of photochemical oxygen depletion during PdTCPP excitation was measured in vivo on the optic disc of piglets by phosphorescence lifetime imaging. Blood-retinal barrier breakdown and tissue damage were assessed by confocal and electron microscopy. RESULTS: For a retinal irradiance of 5 mW/cm(2) at 532 nm and an injected PdTCPP dose of 20 mg/kg, the mean phosphorescence lifetime measured at the optic disc increased from 100 to 600 micros within 8 minutes of continuous illumination. This corresponds to a decrease of pO(2) from 25 to 0 mm Hg, induced by a light dose of only 2.4 J/cm(2). An exposure time of 6 minutes (1.8 J/cm(2)) generated an increase in phosphorescence lifetime from 100 to 400 micros, corresponding to a decrease in pO(2) from 25 to 4 mm Hg. This caused edema in all retinal layers, whereas irradiation of 2 minutes (0.6 J/cm(2)) damaged blood vessels and induced edema in the inner nuclear layer only. Heavy redistribution of occludin occurred after a 30-minute exposure time (9 J/cm(2)). CONCLUSIONS: PdTCPP is potentially phototoxic under certain experimental conditions and can induce damage in peripapillary retina and optic nerve head after light exposure. The severity of tissue damage correlates with the phosphorescence measurements.