Bright, photostable, and nontoxic fluorescent contrast agents are critical for biological imaging. “Self-healing” dyes, in which triplet states are intramolecularly quenched, enable fluorescence imaging by increasing fluorophore brightness and longevity, while simultaneously reducing the generation of reactive oxygen species that promote phototoxicity. Here, we systematically examine the self-healing mechanism in cyanine-class organic fluorophores spanning the visible spectrum. We show that the Baird aromatic triplet-state energy of cyclooctatetraene can be physically altered to achieve order of magnitude enhancements in fluorophore brightness and signal-to-noise ratio in both the presence and absence of oxygen. We leverage these advances to achieve direct measurements of large-scale conformational dynamics within single molecules at submillisecond resolution using wide-field illumination and camera-based detection methods. These findings demonstrate the capacity to image functionally relevant conformational processes in biological systems in the kilohertz regime at physiological oxygen concentrations and shed important light on the multivariate parameters critical to self-healing organic fluorophore design.

明亮、光稳定和无毒的荧光造影剂对于生物成像至关重要。“自我修复”染料，其中三重态在分子内猝灭，通过增加荧光团的亮度和寿命来实现荧光成像，同时减少促进光毒性的活性氧的产生。在这里，我们系统地研究了跨越可见光谱的花青类有机荧光团的自愈机制。我们表明，可以物理改变环辛四烯的 Baird 芳香三重态能量，以在存在和不存在氧气的情况下实现荧光团亮度和信噪比的数量级增强。我们利用这些进步，使用广域照明和基于相机的检测方法，以亚毫秒分辨率直接测量单个分子内的大规模构象动力学。这些发现证明了在生理氧浓度下在千赫兹范围内对生物系统中功能相关的构象过程进行成像的能力，并揭示了对自愈有机荧光团设计至关重要的多变量参数。