Cryo-focused ion beam milling of frozen-hydrated cells has recently provided unprecedented insights into the inner space of cells. In combination with cryo-electron tomography, this method allows access to native structures deep inside cells, enabling structural studies of macromolecules in situ. However, this approach has been mainly limited to individual cells that can be completely vitrified by plunge-freezing. Here, we describe a preparation method that is based on the targeted extraction of material from high-pressure-frozen bulk specimens with a cryo-gripper tool. This lift-out technique enables cryo-electron tomography to be performed on multicellular organisms and tissue, extending the range of applications for in situ structural biology. We demonstrate the potential of the lift-out technique with a structural study of cytosolic 80S ribosomes in a Caenorhabditis elegans worm. The preparation quality allowed for subtomogram analysis with sufficient resolution to distinguish individual ribosomal translocation states and revealed significant cell-to-cell variation in ribosome structure.

冷冻水合细胞的低温聚焦离子束铣削近来为细胞内部空间提供了空前的见识。结合低温电子断层扫描，该方法可以访问细胞内部深处的天然结构，从而可以对大分子进行原位研究。但是，该方法主要限于可通过骤冷完全玻璃化的单个细胞。在这里，我们描述了一种制备方法，该方法基于使用冷冻夹钳工具从高压冷冻的大块标本中有针对性地提取材料的方法。这种提升技术使低温电子断层扫描能够在多细胞生物和组织上进行，从而扩展了原位结构生物学的应用范围。秀丽隐杆线虫蠕虫。制备质量可用于子图分析，并具有足够的分辨率以区分单个核糖体易位状态，并揭示了核糖体结构中细胞间的显着变化。