Fluence-rate effects upon m-THPC photobleaching in a formalin-fixed cell system

We have applied a micro-spectroscopic technique in order to record the laser-induced fluorescence emission of the PDT photosensitiser m-THPC (Foscan) from micron-scale locations within individual formalin-fixed keratinocytes. We demonstrate that m-THPC is highly photolabile in this cellular environment, and that the process of photobleaching can be monitored via the depletion in fluorescence emission during continuous irradiation with 410nm laser light. The progressive reduction of the characteristic 652nm m-THPC fluorescence peak can be described with bi-exponential decay kinetics, consistent with a singlet oxygen-mediated process. The rate of photobleaching, when plotted as a function of light dose, shows inverse fluence-rate dependence. Specifically, the rate of photobleaching induced by the higher laser powers appears to be limited by oxygen availability, as demonstrated by an increase in the (1/e) bleaching dose. Fractionated irradiation provides evidence of intracellular re-oxygenation. These results are in qualitative agreement with previous in vitro and in vivo studies, which indicate that the photodynamic dose delivered during light irradiation is critically dependent upon local fluence rate and oxygen partial pressure.