Ultrastable organic fluorophores for single-molecule research: part II
Single-molecule Förster resonance energy transfer (smFRET) imaging is an integral tool for examining conformational and compositional processes critical to the functions of biological systems. Robustness of smFRET imaging and reproducibility of smFRET efficiencies are increased through photostabilization of fluorophores, state-of-the-art instrumentation, data analysis, and stringent data correction procedures [1,2]. Here, we report that small molecule photostabilizing agents in imaging buffers are although important for photostabilization of fluorophores, they can be bottlenecks for quantitative smFRET imaging due to their varied photophysical and photochemical impacts on fluorophores. To mitigate these problems, we provide a simple strategy of using ‘self-healing’ organic fluorophores that are intramolecularly photostabilized by cyclooctatetraene (COT) as a triplet state quencher. COT-mediated self-healing mechanism enables robust determination of true smFRET efficiency of organic fluorophores on both nucleic acids and proteins in oxygenated as well as in deoxygenated imaging buffers. These findings conceptually advance our understanding of smFRET and provide strategies for robust, quantitative smFRET imaging .
 Hellenkamp et al., Nat. Methods, 15, 669–676 (2018).
 Lerner et al., eLife, 10:e60416 (2021).
 Pati et al., manuscript to be submitted.