Fluorescence Photobleaching and Photoproduct Formation

Sensitizer fluorescence photobleaching and photoproduct accumulation during PDT

Most photosensitizers used in photodynamic therapy are chemically modified by the photochemistry that they initiate. These modifications may take the form of photoproducts with fluorescence spectra that differ from that of the parent compound, and/or the irreversible photobleaching of the sensitizer, which gives rise to a progressive loss of fluorescence during irradiation. Thus, these modifications in fluorescence report something about the local photochemistry in the cells or tissue undergoing PDT. For this reason, monitoring sensitizer photobleaching and photoproduct accumulation during treatment has received some attention as a possible surrogate reporter of photodynamic dose deposition and biological response. We have been very active in this area during the past several years. In particular, we have been interested in the irradiance dependence of photobleaching/photoproduct formation because in a number of model systems the biological response to PDT shows a strong such dependence.

Spectra acquired in an intact spheroid incubated with h-ALA for 3 hours. Contributions from PpIX and photoproducts are shown for spectra acquired pre- and post-PDT in different regions of the spheroid.

Our work has resulted in several recent publications (see Group Publications) including those of J.C. Finlay et al., 2001; 2002, where we reported on interesting irradiance dependent features of the fluorescence emission spectra of ALA-induced protoporphyrin IX and mTHPC, respectively. Similar work with Photofrin is being done now and will be reported in the near future. The paper by S. Coutier et al. (2001) describes experiments and analysis performed in collaboration with a group at the Centre Alexis Vautrin in Nancy, France. That work established a relationship between the extent of mTHPC bleaching and clonogenic survival in a multicell tumor spheroid model. We are currently using confocal imaging and spectroscopy to evaluate the spatial patterns of bleaching and photoproduct formation in optical sections of intact spheroids in order to confirm interpretations of the spectral changes identified in vivo.