Section Electron Microscopy, Dept. of Cell and Chemical Biology, Leiden University Medical Center, 2300 RC, Leiden, The Netherlands.
NeCEN, Gorlaeus Laboratories, Leiden University, 2333 CC, Leiden, The Netherlands.
Sci Rep. 2019 Feb 4;9(1):1369. doi: 10.1038/s41598-018-37728-8.
Sample fixation by vitrification is critical for the optimal structural preservation of biomolecules and subsequent high-resolution imaging by cryo-correlative light and electron microscopy (cryoCLEM). There is a large resolution gap between cryo fluorescence microscopy (cryoFLM), ~400-nm, and the sub-nanometre resolution achievable with cryo-electron microscopy (cryoEM), which hinders interpretation of cryoCLEM data. Here, we present a general approach to increase the resolution of cryoFLM using cryo-super-resolution (cryoSR) microscopy that is compatible with successive cryoEM investigation in the same region. We determined imaging parameters to avoid devitrification of the cryosamples without the necessity for cryoprotectants. Next, we examined the applicability of various fluorescent proteins (FPs) for single-molecule localisation cryoSR microscopy and found that all investigated FPs display reversible photoswitchable behaviour, and demonstrated cryoSR on lipid nanotubes labelled with rsEGFP2 and rsFastLime. Finally, we performed SR-cryoCLEM on mammalian cells expressing microtubule-associated protein-2 fused to rsEGFP2 and performed 3D cryo-electron tomography on the localised areas. The method we describe exclusively uses commercially available equipment to achieve a localisation precision of 30-nm. Furthermore, all investigated FPs displayed behaviour compatible with cryoSR microscopy, making this technique broadly available without requiring specialised equipment and will improve the applicability of this emerging technique for cellular and structural biology.
玻璃化固定对于生物分子的最佳结构保存以及随后通过冷冻相关的光和电子显微镜（cryoCLEM）进行高分辨率成像至关重要。冷冻荧光显微镜（cryoFLM）的分辨率约为 400nm，而冷冻电子显微镜（cryoEM）可达到亚纳米分辨率，这两者之间存在较大的分辨率差距，这阻碍了 cryoCLEM 数据的解释。在这里，我们提出了一种使用 cryo 超分辨率（cryoSR）显微镜提高 cryoFLM 分辨率的通用方法，该方法与同一区域内连续的 cryoEM 研究兼容。我们确定了成像参数，以避免 cryosample 的非晶化，而无需使用 cryoprotectants。接下来，我们研究了各种荧光蛋白（FP）在单分子定位 cryoSR 显微镜中的适用性，发现所有研究的 FP 都显示出可逆的光可切换行为，并在标记有 rsEGFP2 和 rsFastLime 的脂质纳米管上证明了 cryoSR。最后，我们对表达微管相关蛋白-2 的融合 rsEGFP2 的哺乳动物细胞进行了 SR-cryoCLEM，并对定位区域进行了 3D 冷冻电子断层扫描。我们描述的方法仅使用市售设备即可实现 30nm 的定位精度。此外，所有研究的 FP 都表现出与 cryoSR 显微镜兼容的行为，这使得该技术在无需专用设备的情况下广泛可用，并将提高该新兴技术在细胞和结构生物学中的适用性。
