Effect of PpIX photoproducts formation on pO2 measurement by time-resolved delayed fluorescence spectroscopy of PpIX in solution and in vivo

Emmanuel Louis Arthur Gerelli, Veronika Huntosova, Georges Wagnières, Matthieu Zellweger
Elsevier Science Sa, 2016
Journal paper

Abstract

The measurement of Protoporphyrin IX delayed fluorescence lifetime is a minimally invasive method for monitoring the levels of oxygen in cells and tissues. The excitation of Protoporphyrin IX during this measurement can lead to the formation of photoproducts in vitro and in vivo. The influence of their luminescence on the measured Protoporphyrin IX delayed fluorescence lifetimes was studied in solution and in vivo on the Chick's chorioallantoic membrane (CAM) model under various oxygen enriched air conditions (0mmHg, 37mmHg and 155mmHg). The presence of photoproducts disturbs such measurements since the delayed fluorescence emission of some of them spectrally overlaps with that of Protoporphyrin IX. One possible way to avoid this obstacle is to detect Protoporphyrin IX's delayed fluorescence lifetime in a very specific spectral range (620-640nm). Another possibility is to excite Protoporphyrin IX with light doses much lower than 10J/cm(2), quite possibly as low as a fraction 1J/cm(2) at 405nm. This leads to an increased accuracy of pO2 detection. Furthermore, this method allows combination of diagnosis and therapy in one step. This helps to improve detection systems and real-time identification of tissue respiration, which is tuned for the detection of PpIX luminescence and not its photoproducts.

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The determination of the oxygen partial pressure (pO(2)) in real time in living biological tissues is of high interest for numerous therapeutics, including photodynamic therapy (PDT) and radiotherapy. The minimally invasive and real-time measurement of the pO(2) also enables to obtain interesting fundamental information regarding the metabolic activities in cells and tissues. The development of time-resolved luminescence measurement (TRLM) methods combined with the availability of new oxygen-sensitive molecular probes is at the origin of the significant progress that have been achieved during these past decades to measure the pO(2) in living organisms. These probes include porphyrins, such as aminolevulinic acid-induced protoporphyrin IX (PPIX), which is an approved photosensitizer. Using the photosensitizer to probe the pO(2) is of high interest in PDT since the level of oxygen is measured at the precise location where the phototoxic mechanisms take place. However, PPIX has drawbacks to measure the pO(2) by TRLM, including its significant photobleaching. Since the PPIX excitation during pO(2) measurements leads to the generation of its photoproducts, we studied the impact of their luminescence on the measurement of the PPIX triplet state lifetime in solution and in vivo on the Chick's Chorioallantoic Membrane (CAM) model. We performed this study under various oxygen conditions. Our results indicate that perturbations induced by these photoproducts can be avoided if the PPIX luminescence is detected between 620 and 640 nm, or if PPIX is excited at 405 nm with light doses < 1 J/cm(2).

Spie-Int Soc Optical Engineering2017

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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. (C) 2011 Society of Photo-Optical Instrumentation Engineers (SPIE). [DOI: 10.1117/1.3558846]

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