Suppressing Autogenic ROS in a Singlet Oxygen Sensor through Triplet-Triplet Energy Transfer

Reactive oxygen species (ROS), particularly singlet oxygen (1O2), serve as critical signaling molecules but their excessive accumulation accelerates cellular aging, necessitating robust detection methods. Singlet oxygen sensor green (SOSG) is a popular fluorescent turn-on probe for 1O2 quantification both in vitro and in vivo. However, SOSG itself generates ROS, including 1O2, under ultraviolet and visible light even without external 1O2 sources. In this work, using fluorescence correlation spectroscopy, we reveal that SOSG forms microsecond-lived triplet dark states, acting as precursors to probe-derived ROS. Triplet state quenchers, such as cyclooctatetraene, working via triplet-triplet energy transfer mechanism, shorten SOSG's triplet lifetime by up to 2-fold, when added at millimolar concentrations in aqueous buffer. This suppression of triplet buildup reduces autogenic ROS formation, a trend further supported by Monte Carlo simulations. Our findings clarify the molecular origins of probe-induced ROS and propose strategies for accurate, quantitative ROS imaging.