Quantitative Monitoring of Subcutaneous Tissue Oxygenation via Phosphorescent Polymer Dots and Time-Gated Lifetime Imaging

Precise quantification of tissue oxygenation (pO 2 ) is important for understanding tumor progression and evaluating metabolic therapies. However, conventional intensity-based optical oxygen sensors suffer from severe signal fluctuations for in vivo measurements due to tissue scattering and excitation conditions. Here, we present a robust quantitative oxygen sensing platform by using ultra-bright phosphorescent semiconducting polymer dots (Pdots) and a custom-built time-gated phosphorescence lifetime imaging (PLIM) system. Polyfluorene serves as a light-harvesting unit that sensitizes oxygen-responsive metalloporphyrin moieties through intraparticle energy transfer, yielding bright oxygen-sensitive Pdots with a large excitation-emission separation. The PLIM system consists of an intensified scientific complementary metal-oxide-semiconductor (sCMOS) camera with an internal trigger for pulsed LED excitation. We demonstrated the longitudinal imaging of local microenvironmental pO 2 in a murine breast tumor model over an 8-day period. While intensity imaging suffers from signal degradation due to tumor growth and probe dilution, the PLIM system reliably captures a dynamic change in tumor oxygenation, with pO 2 increasing from a hypoxic level (<10 mmHg) on Day 1 to ~43 mmHg on Day 8. This trend likely reflects tumor vascular remodeling during early-stage growth. Our study provides a promising tool for tracking subcutaneous tissue metabolic dynamics and evaluating hypoxia-targeted cancer therapies.