Insights into photodynamic therapy dosimetry: simultaneous singlet oxygen luminescence and photosensitizer photobleaching measurements.

Photodynamic therapy (PDT) is generally based on the generation of highly reactive singlet oxygen ((1)O(2)) through interactions of photosensitizer, light, and oxygen ((3)O(2)). These three components are highly interdependent and dynamic, resulting in variable temporal and spatial (1)O(2) dose deposition. Robust dosimetry that accounts for this complexity could improve treatment outcomes. Although the 1270 nm luminescence emission from (1)O(2) provides a direct and predictive PDT dose metric, it may not be clinically practical. We used (1)O(2) luminescence (or singlet oxygen luminescence (SOL)) as a gold-standard metric to evaluate potentially more clinically feasible dosimetry based on photosensitizer bleaching. We performed in vitro dose-response studies with simultaneous SOL and photosensitizer fluorescence measurements under various conditions, including variable (3)O(2), using the photosensitizer meta-tetra(hydroxyphenyl)chlorin (mTHPC). The results show that SOL was always predictive of cytotoxicity and immune to PDT's complex dynamics, whereas photobleaching-based dosimetry failed under hypoxic conditions. However, we identified a previously unreported 613 nm emission from mTHPC that indicates critically low (3)O(2) levels and can be used to salvage photobleaching-based dosimetry. These studies improve our understanding of PDT processes, demonstrate that SOL is a valuable gold-standard dose metric, and show that when used judiciously, photobleaching can serve as a surrogate for (1)O(2) dose.