Reflectance and fluorescence spectroscopies in photodynamic therapy

In vivo fluorescence spectroscopy during photodynamic therapy (PDT) has the potential to provide information on the distribution and degradation of sensitizers, the formation of fluorescent photoproducts and changes in tissue autofluorescence induced by photodynamic treatment. Reflectance spectroscopy allows quantification of light absorption and scattering in tissue. We present the results of several related studies of fluorescence and reflectance spectroscopy and their applications to photodynamic dosimetry. First, we develop and test an empirical method for the correction of the distortions imposed on fluorescence spectra by absorption and scattering in turbid media. We characterize the irradiance dependence of the in vivo photobleaching of three sensitizers, protoporphyrin IX (PpIX), Photofrin and mTHPC, in a rat skin model. The photobleaching and photoproduct formation of PpIX exhibit irradiance dependence consistent with singlet oxygen (O2)-mediated bleaching. The bleaching of mTHPC occurs in two phases, only one of which is consistent with a O2-mediated mechanism. Photofrin’s bleaching is independent of irradiance, although its photoproduct formation is not. This can be explained by a mixed-mechanism bleaching model. Second, we develop an algorithm for the determination of tissue optical prop-