Evaluation of a time-resolved singlet oxygen detection system for in vivo photodynamic therapy

Singlet oxygen (¹O₂) is the primary cytotoxic agent in Type-II photodynamic therapy (PDT). Single-photon avalanche diode (SPAD)-based time-resolved singlet oxygen luminescence detection (TSOLD) systems enable direct detection of the near-infrared ¹O₂ phosphorescence (∼1270 nm) during PDT, offering a powerful tool for treatment monitoring. However, the efficiency of detection depends strongly on the acquisition parameters. Here, we present a comprehensive evaluation of a TSOLD system with 630 nm and 690 nm nanosecond pulsed diode lasers developed for in vivo PDT monitoring. The influence of acquisition time (1-300 s), SPAD dead time (5-40 µs), and temporal bin width (1-100 ns) on the fidelity of ¹O₂ lifetime measurements and signal-to-noise ratio (SNR) was quantitatively investigated. Measurements were performed using liquid phantoms containing the photosensitizers Photofrin or benzoporphyrin derivative, with acquisition time further validated in vivo in murine tumor models. The results demonstrate that both acquisition time and detector dead time significantly affect the signal vs. time histogram shape and the accuracy of the ¹O₂ and photosensitizer triplet-state lifetime measurements, while an optimal bin width minimizes photon-count noise without compromising temporal resolution. The optimized parameters enable reliable ¹O₂ lifetime extraction within 100 s from the mouse model in vivo. This systematic evaluation establishes quantitative design guidelines for compact TSOLD systems tailored to in vivo applications.