Department of Chemistry , Stanford University , Stanford , California 94305 , United States.
Department of Developmental Biology , Stanford University School of Medicine , Stanford , California 94305 , United States.
J Am Chem Soc. 2018 Oct 3;140(39):12310-12313. doi: 10.1021/jacs.8b05960. Epub 2018 Sep 20.
Single-molecule super-resolution fluorescence microscopy conducted in vitrified samples at cryogenic temperatures offers enhanced localization precision due to reduced photobleaching rates, a chemical-free and rapid fixation method, and the potential of correlation with cryogenic electron microscopy. Achieving cryogenic super-resolution microscopy requires the ability to control the sparsity of emissive labels at cryogenic temperatures. Obtaining this control presents a key challenge for the development of this technique. In this work, we identify a red photoactivatable protein, PAmKate, which remains activatable at cryogenic temperatures. We characterize its activation as a function of temperature and find that activation is efficient at cryogenic and room temperatures. We perform cryogenic super-resolution experiments in situ, labeling PopZ, a protein known to assemble into a microdomain at the poles of the model bacterium Caulobacter crescentus. We find improved localization precision at cryogenic temperatures compared to room temperature by a factor of 4, attributable to reduced photobleaching.
在低温下进行的玻璃化冷冻样品的单分子超分辨率荧光显微镜技术，由于降低了光漂白率、采用了无化学试剂的快速固定方法，以及与低温电子显微镜相关联的潜力，提供了更高的定位精度。实现低温超分辨率显微镜需要能够在低温下控制发射标记的稀疏度。获得这种控制对于该技术的发展提出了一个关键的挑战。在这项工作中，我们鉴定了一种红色光可激活蛋白 PAmKate，它在低温下仍然可以被激活。我们研究了其激活与温度的关系，并发现其在低温和室温下都具有高效的激活能力。我们在原位进行了低温超分辨率实验，标记了 PopZ，这是一种已知在模型细菌新月柄杆菌的两极组装成微区的蛋白质。与室温相比，我们发现低温下的定位精度提高了 4 倍，这归因于光漂白的减少。
