Predicting the therapeutic outcome of photodynamic therapy (PDT) requires knowledge of the amount of cytoxic species generated. An implicit approach to assessing PDT efficacy has been proposed where changes in photosensitizer (PS) fluorescence during treatment are used to predict treatment outcome. To investigate this,
in vitroexperiments were performed in which Mat‐LyLu cells were incubated in meta‐tetra(hydroxyphenyl)chlorin (mTHPC) and then irradiated with 652 nm light. PS concentration, fluence rate and oxygenation were independently controlled and monitored during the treatment. Fluorescence of mTHPC was monitored during treatment and, at selected fluence levels, cell viability was determined using a colony‐formation assay. Singlet oxygen dose was calculated using four different models and was compared with cell survival. For the dose metric based on singlet oxygen–mediated PS photobleaching, a universal relationship between cell survival and singlet oxygen dose was found for all treatment parameters. Analysis of the concentration dependence of bleaching suggests that the lifetime of singlet oxygen within the cell is 0.05–0.25 μs. Generation of about 9 × 108 molecules of singlet oxygen per cell reduces the surviving fraction by 1/e.