Photophysical performances of organic fluorophores in modern imaging applications, including single-molecule and super-resolution fluorescence microscopy, are compromised by their transient excursions to dark triplet and radical states causing stochastic photo blinking and irreversible photobleaching. To circumvent these problems, we develop and study âself-healingâ organic fluorophores, in which the dark triplet states are intramolecularly quenched by triplet state quenchers [1,2]. This intramolecular photostabilization approach dramatically increases fluorophore brightness, signal-to-noise ratio, and photostability, while simultaneously reduces phototoxicity by decreasing the generation of reaction oxygen species. The performance enhancements of the fluorophores enable us to achieve robust, submillisecond recordings of protein dynamics using wide-field illumination and camera-based single-molecule Förster resonance energy transfer (smFRET) techniques reaching the theoretical speed limit of camera-based detections [3]. These findings extend the potential to image single molecules in vitro and in live-cell applications in the absence of solution-based photostabilizers at physiological oxygen concentrations in the kilohertz regime [3,4] and shed important light on the multivariate parameters critical to self-healing organic fluorophore design for biological research.
[1] Altman et al., Nat. Methods, 9, 68â71 (2011).
[2] Zheng et al., Chem. Soc. Rev., 43, 1044â1056 (2014).
[3] Pati et al., PNAS, 117, 24305â24315 (2020).
[4] Asher et al., Nat. Methods, 18, 397â405 (2021).