Department of Chemistry, Stanford University, Stanford, California 94305, United States.
Biosciences Division, SLAC National Accelerator Laboratory, Menlo Park, California 94025, United States.
J Am Chem Soc. 2024 Oct 23;146(42):28707-28716. doi: 10.1021/jacs.4c05632. Epub 2024 Oct 10.
Super-resolved cryogenic correlative light and electron microscopy is a powerful approach which combines the single-molecule specificity and sensitivity of fluorescence imaging with the nanoscale resolution of cryogenic electron tomography. Key to this method is active control over the emissive state of fluorescent labels to ensure sufficient sparsity to localize individual emitters. Recent work has identified fluorescent proteins (FPs) that photoactivate or photoswitch efficiently at cryogenic temperatures, but long on-times due to reduced quantum yield of photobleaching remain a challenge for imaging structures with a high density of localizations. In this work, we explore the photophysical properties of the red photoactivatable FP PAmKate and identify a 2-color process leading to enhanced turn-off of active emitters, improving localization rate. Specifically, after excitation of ground state molecules, we find that a transient state forms with a lifetime of ∼2 ms under cryogenic conditions, which can be bleached by exposure to a second wavelength. We measure the response of the transient state to different wavelengths, demonstrate how this mechanism can be used to improve imaging, and provide a blueprint for the study of other FPs at cryogenic temperatures.
超分辨低温相关光学和电子显微镜是一种强大的方法，它将荧光成像的单分子特异性和灵敏度与低温电子断层扫描的纳米分辨率相结合。该方法的关键是主动控制荧光标记的发射状态，以确保足够的稀疏性来定位单个发射器。最近的工作已经确定了在低温下有效光激活或光开关的荧光蛋白 (FP)，但由于光漂白量子产率降低，长时间的光激活仍然是对具有高密度定位结构进行成像的一个挑战。在这项工作中，我们探索了红色光可激活 FP PAmKate 的光物理性质，并确定了一种导致活性发射器更有效关闭的双色过程，从而提高了定位率。具体来说，在激发基态分子后，我们发现在低温条件下形成了一种寿命约为 2 毫秒的瞬态，该瞬态可以通过暴露在第二个波长下被漂白。我们测量了瞬态对不同波长的响应，演示了这种机制如何用于改善成像，并为在低温下研究其他 FP 提供了蓝图。
