Time-domain luminescence lifetime imaging with oxygen sensor particles in the presence of actinic light

Time-gated luminescence lifetime imaging is highly valued in quantitative chemical imaging for its robustness against inhomogeneities in signal intensity and especially background fluorescence, amongst other things. While some literature describes the presence of actinic light during time-gated imaging as problematic, the performance of time-gated imaging in the presence of broadband actinic light remains largely unexplored. To close this knowledge gap, we systematically investigating the influence of broadband actinic light in optical O₂ imaging using water dispersible NIR-emitting O₂ sensor particles with a new open-source, luminescence lifetime imaging system. Mean lifetimes measured via time-gated lifetime imaging with properly applied background subtraction showed no influence of actinic light, even at high photon irradiance levels corresponding to mid-day full solar irradiation. Such measurements also showed no sensitivity to background fluorescence with or without actinic light, as long as the background light remained constant in the timeframe of the measurement (∼100 ms). We demonstrate the capability of the imaging system for imaging O₂ dynamics during experimental light-dark shifts in biofilms and for intra-tissue O₂ measurements in living corals. The realization of O₂ imaging even under strong actinic light levels, now enables a wide range of applications in environmental, biotechnological and biomedical studies, where beneficial or harmful effects of light treatment are investigated.