The diagnostics depth of tumour during photodynamic diagnosis (PDD) is dependent on the wavelength of the excitation light, as there is the attenuation of light by absorption and scattering. The use of 505 nm excitation to extend the diagnostics depth of deeply located tumour has been reported. However, the fluorescence emission intensity during fluorescence observation of the tumour is affected by the photobleaching of the photosensitizer. The photosensitizer photobleaching is accompanied by the formation of its photoproduct. This study has investigated the potential use of fluorescence emission from protoporphyrin IX (PpIX), followed by the combined use of the fluorescence emission from PpIX and its photoproduct, for the fluorescence observation of deeply located tumours. We have introduced the concept of fluorescence photoswitching, where the excitation of the photoproduct formed during the initial PDD, can lead to fluorescence intensity higher than that obtained by PpIX excitation. An increase in the fluorescence detection intensity and time for tumour detection may be achieved with fluorescence photoswitching. Preliminary experiments on fluorescence photoswitching were performed for PpIX in solution and ex vivo. Fluorescence photoswitching was observed in both forms of PpIX investigated, with a maximum of 94 % initial PpIX intensity obtained with the excitation of the photoproduct. It is expected that a higher fluorescence emission can be attained with the optimisation of the irradiation conditions. Although further investigations are required, this work has demonstrated the potential of fluorescence photoswitching for extending the fluorescence observation time for the PDD of deeply located tumours.
SignificancePhotobleaching of the photosensitizer reduces fluorescence observation time and the intensity of fluorescence emitted for tumor detection during 5-aminolevulinic acid-based photodynamic diagnosis.AimThis 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.ApproachThe 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.ResultsFluorescence 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.ConclusionsWe 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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