In Vivo Assessment of Benzoporphyrin Uptake and Singlet Oxygen Generation in Mice for Photodynamic Therapy Monitoring

The efficacy of photodynamic therapy (PDT) is strongly influenced by the biodistribution of the photosensitizer and the local generation of ¹O₂ within tumor tissue. However, real-time in vivo monitoring of these critical parameters for clinically approved photosensitizers remain a major challenge in translational photodynamic research. We report a novel portable bifurcated fiber-coupled time-resolved singlet oxygen luminescence detection instrument combining an integrating pulsed 690 nm diode laser system. Using this instrument, ¹O₂ signal is estimated corresponding to the uptake kinetics of the clinical photosensitizer benzoporphyrin derivative (BPD) in tumor-bearing mice up to 3 h post injection along with pre- and post-PDT ¹O₂ luminescence were measured in the same mice. The measured ¹O₂ counts correlate with the increase in BPD accumulation in the tumor region from 15 min to 2 h post injection and then remain constant in the 2-3 h measurement period. A strong positive correlation was observed between local BPD uptake and singlet oxygen signal. The estimated lifetime of ¹O₂ in vivo was 0.25-0.35 μs. The TSOLD system provided consistent, noninvasive readouts of ¹O₂ generation in real time with minimal background interference. Control experiments using BPD-free conditions confirmed the specificity of the detected signal. This study demonstrates a novel, noninvasive optical approach for simultaneous murine in vivo quantification of photosensitizer uptake and singlet oxygen production during PDT. This portable TSOLD instrument enables dynamic monitoring of therapeutic conditions in preclinical cancer models and has potential for future adaptation to clinical settings, supporting more precise and personalized PDT planning and dosimetry.