The application of cryo-correlative light and cryo-electron microscopy (cryo-CLEM) gives us a way to locate structures of interest in the electron microscope. In brief, the structures of interest are fluorescently tagged, and images from the cryo-fluorescent microscope (cryo-FM) maps are superimposed on those from the cryo-electron microscope (cryo-EM). By enhancing cryo-FM to include single-molecule localization microscopy (SMLM), we can achieve much better localization. The introduction of cryo-SMLM increased the yield of photons from fluorophores, which can benefit localization efforts. Dahlberg and Moerner (2021, Annual Review of Physical Chemistry, 72, 253–278) have a recent broad and elegant review of super-resolution cryo-CLEM. This paper focuses on cryo(F)PALM/STORM for the cryo-electron tomography community. I explore the current challenges to increase the accuracy of localization by SMLM and the mapping of those positions onto cryo-EM images and maps. There is much to consider: we need to know if the excitation of fluorophores damages the structures we seek to visualize. We need to determine if higher numerical aperture (NA) objectives, which add complexity to image analysis but increase resolution and the efficiency of photon collection, are better than lower NA objectives, which pose fewer problems. We need to figure out the best way to determine the axial position of fluorophores. We need to have better ways of aligning maps determined by FM with those determined by EM. We need to improve the instrumentation to be easier to use, more accurate, and ice-contamination free. The bottom line is that we have more work to do.

中文翻译：

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我的蛋白质在细胞的什么地方？

低温相关光和低温电子显微镜 (cryo-CLEM) 的应用为我们提供了一种在电子显微镜中定位感兴趣结构的方法。简而言之，感兴趣的结构被荧光标记，来自低温荧光显微镜 (cryo-FM) 图的图像叠加在来自低温电子显微镜 (cryo-EM) 的图像上。通过增强 cryo-FM 以包括单分子定位显微镜 (SMLM)，我们可以实现更好的定位。低温 SMLM 的引入增加了荧光团的光子产量，这有利于本地化工作。Dahlberg 和 Moerner (2021, 物理化学年度回顾,72, 253–278) 最近对超分辨率冷冻 CLEM 进行了广泛而优雅的评论。本文重点介绍低温电子断层扫描领域的低温（F）PALM/STORM。我探讨了当前挑战，以提高 SMLM 定位的准确性以及将这些位置映射到冷冻 EM 图像和地图上。有很多事情需要考虑：我们需要知道荧光团的激发是否会损坏我们试图可视化的结构。我们需要确定较高的数值孔径 (NA) 物镜是否比低数值孔径 (NA) 物镜更好，后者会增加图像分析的复杂性，但会提高分辨率和光子收集的效率，而较低的数值孔径 (NA) 物镜会带来较少的问题。我们需要找出确定荧光团轴向位置的最佳方法。我们需要有更好的方法将 FM 确定的地图与 EM 确定的地图对齐。我们需要改进仪器，使其更易于使用、更准确且无冰污染。最重要的是，我们还有更多工作要做。