Photobleaching and photoproduct formations are considered essential phenomena in improving the efficacy of photodynamic diagnosis and therapy (PDD and PDT). We investigated the photobleaching of protoporphyrin IX (PpIX) by measuring its concentration with mass spectrometry (MS). The reduction in the concentration of PpIX dissolved in dimethyl sulfoxide was measured during PDD and PDT conditions using lasers with wavelengths of 405 and 635 nm, respectively, at a power density of 10, 50 or 100 mW/cm2. The obtained results were compared with the results of conventional fluorescence spectroscopy and previously reported results. Our results demonstrate the variation in the MS‐based photobleaching coefficient of PpIX with the power density, while the fluorescence‐based photobleaching coefficient was independent of the power density. The results of MS also show faster photobleaching of PpIX in comparison with that obtained from fluorescence. The difference may be attributed to the change in the fluorescence quantum yield of PpIX with its concentration and the effect of fluorescence emission from the PpIX photoproducts. Thus, an MS‐based investigation of the photobleaching poses to be a more stable investigation form. Our finding highlights the importance of recognizing the potential significance of these discoveries in the PDD and PDT dosimetry and efficacy.
Significance: Photobleaching of the photosensitizer reduces fluorescence observation time and the intensity of fluorescence emitted for tumor detection during 5-aminolevulinic acid-based photodynamic diagnosis.Aim: This study aims to utilize the concept of fluorescence photoswitching, which uses the fluorescence emission from photosensitizer excitation followed by the simultaneous excitation of the photosensitizer and its photoproduct to increase the fluorescence detection intensity during PDD of deeply located tumors. Approach:The fluorescence photobleaching of protoporphyrin IX (PpIX) and the formation of its photoproduct, photoprotoporhyrin (Ppp), caused by exposure to 505 nm light were investigated in solution, ex vivo, and in vivo, and the fluorescence photoswitching was analyzed. The fluorescence observations of PpIX and Ppp were performed with 505 and 450 or 455 nm excitation, respectively, which is the suited wavelength for the primary excitation of each fluorophore.Results: Fluorescence photoswitching was observed in all forms of PpIX investigated, and the fluorescence photoswitching time, fluorescence intensity relative to the initial PpIX and Ppp intensity, and fluorescence intensity relative to PpIX after photobleaching were obtained. The dependence of the fluorescence photoswitching time and intensity on the irradiation power density was noted. A fluorescence intensity increase between 1.6 and 3.9 times was achieved with simultaneous excitation of PpIX and Ppp after fluorescence photoswitching, compared with the excitation of PpIX alone. Conclusions:We have demonstrated the potential of fluorescence photoswitching for the improvement of the fluorescence observation intensity for the PDD of deeply located tumors.
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