Membrane biology of viral infection laboratory, Schaller Research Groups, Department of Infectious Diseases, Virology, Heidelberg University Hospital, Heidelberg, Germany.
Membrane biology of viral infection laboratory, Schaller Research Groups, Department of Infectious Diseases, Virology, Heidelberg University Hospital, Heidelberg, Germany.
Methods Cell Biol. 2021;162:273-302. doi: 10.1016/bs.mcb.2020.12.009. Epub 2021 Feb 17.
In situ cryo-electron tomography of cryo-focused ion beam (cryo-FIB) milled cells enables the study of cellular organelles in unperturbed conditions and close to the molecular resolution. However, due to the crowdedness of the cellular environment, the identification of individual macromolecular complexes either on organelles or inside the cytosol in cryo-electron tomograms is challenging. Cryo-correlative light and electron microscopy (cryo-CLEM) employs a fluorescently labeled feature of interest imaged by cryo-light microscopy that is correlated to cryo-electron microscopy maps of cryo-FIB milled lamellae using correlation markers discernable by both imaging methods. Here, we provide a protocol for a post-correlation on-lamella cryo-CLEM approach for localization of fluorescently labeled organelles of interest in cryo-lamellae after cryo-FIB milling and tomography of adherent plunge frozen cells.
在原位冷冻聚焦离子束（cryo-FIB）铣削细胞的冷冻电子断层扫描使人们能够在未受干扰的条件下并接近分子分辨率研究细胞细胞器。然而，由于细胞环境的拥挤，在冷冻电子断层扫描中识别细胞器上或细胞溶胶内的单个大分子复合物具有挑战性。冷冻相关的光和电子显微镜（cryo-CLEM）采用通过 cryo-光显微镜成像的感兴趣的荧光标记特征，该特征与使用两种成像方法可辨别的相关标记物相关联，与 cryo-FIB 铣削薄片的 cryo 电子显微镜图谱相关联。在这里，我们提供了一种在关联后在薄片上进行 cryo-CLEM 的方法，用于在 cryo-FIB 铣削和贴壁式急冻细胞的断层扫描之后，对 cryo 薄片中荧光标记的感兴趣的细胞器进行定位。
