Optimizing excitation wavelength for enhanced singlet oxygen generation with benzoporphyrin derivatives in photodynamic therapy

Detection of singlet oxygen (1O2) generation during photodynamic therapy (PDT) remains a challenge, especially in vivo, due to the extremely weak signal and interference of background emissions. We developed a bifurcated, fiber-based system for time-resolved singlet oxygen luminescence (TSOLD) to detect 1O2in vivo. The system integrates a pulsed diode laser, a bifurcated fiber optic, a single-photon avalanche diode (SPAD), customized long and bandpass filters, and time-tagger electronics. The bifurcated system design facilitates the physical separation of detection and excitation channels, thereby minimizing crosstalk and enhancing the signal-to-noise ratio. This configuration allows for the simultaneous collection of both sub-surface and surface signals in small animal models. To ensure the validity of the system, we measured singlet oxygen with benzoporphyrin derivative (BPD) in ethanol with and without sodium azide. Further, we analyse the cumulative 1O2generation by BPD in liquid phantoms and C3H mice excited by a pulse diode laser at the following wavelengths: 405, 520, 640, and 690nm. Photon-counting measurements of 1O2luminescence at 1270nm reveal that after excitation with a 690nm pulsed laser, BPD can produce significantly higher signals of 1O2as compared to other wavelengths in liquid phantoms. In an in vivo study, the 690nm laser demonstrated a capacity to generate 2 to 3 times more singlet oxygen in C3H mice as compared to other excitation wavelengths. This enhanced 1O2production is attributable to superior tissue penetration and the effective absorption of benzoporphyrin derivative (BPD) at 690nm excitation wavelength. This bifurcated-fiber TSOLD system represents a significant advancement in 1O2detection technology, enabling real-time, minimally invasive monitoring during PDT. It supports preclinical assessments, optimizes PDT light delivery planning, and facilitates the clinical translation of 1O2based dosimetry.