In vivo measurement of the tissue oxygenation by time-resolved luminescence spectroscopy of protoporphyrin IX: Strategies to minimize artefacts associated with photoproducts

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).