Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins
Correlative cryo super-resolution light and electron microscopy on mammalian cells using fluorescent proteins
复制标题
DOI：
10.1038/s41598-018-37728-8
复制
发表时间：
2019-02-04
影响因子：
4.6
通讯作者：
Sharp, Thomas H.
中科院分区：
文献类型：
--
作者：
Tuijtel, Maarten W.;Koster, Abraham J.;Sharp, Thomas H.
通过玻璃化固定样品对于生物分子的最佳结构保存和随后的低温相关光学和电子显微镜（cryoCLEM）的高分辨率成像至关重要。低温荧光显微镜（cryoFLM）的分辨率类似于400纳米，与低温电子显微镜（cryoEM）的亚纳米分辨率之间存在很大的分辨率差距，这阻碍了对cryoCLEM数据的解释。在这里，我们提出了一种通用的方法来提高低温超分辨率（cryoSR）显微镜的分辨率，这与在同一区域的连续低温电镜研究是兼容的。我们确定了成像参数，以避免在不需要冷冻保护剂的情况下冷冻样品的脱玻化。接下来，我们检查了各种荧光蛋白（FPs）在单分子定位cryoSR显微镜中的适用性，发现所有研究的FPs都表现出可逆的光开关行为，并在用rsEGFP2和rsFastLime标记的脂质纳米管上展示了cryoSR。最后，我们对表达微管相关蛋白-2与rsEGFP2融合的哺乳动物细胞进行了SR-cryoCLEM，并对局部区域进行了3D低温电子断层扫描。我们描述的方法专门使用市售设备来实现30纳米的定位精度。此外，所有研究的FPs都显示出与cryoSR显微镜兼容的行为，使该技术在不需要专门设备的情况下广泛可用，并将提高这种新兴技术在细胞和结构生物学中的适用性。
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), similar to 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.