Cryo-electron tomography (cryo-ET) provides a promising approach to study intact structures of macromolecules in situ, but the efficient preparation of high-quality cryosections represents a bottleneck. Although cryo-focused ion beam (cryo-FIB) milling has emerged for large and flat cryo-lamella preparation, its application to tissue specimens remains challenging. Here, we report an integrated workflow, VHUT-cryo-FIB, for efficiently preparing frozen hydrated tissue lamella that can be readily used in subsequent cryo-ET studies. The workflow includes vibratome slicing, high-pressure freezing, ultramicrotome cryo-trimming and cryo-FIB milling. Two strategies were developed for loading cryo-lamella via a side-entry cryo-holder or an FEI AutoGrid. The workflow was validated by using various tissue specimens, including rat skeletal muscle, rat liver and spinach leaf specimens, and in situ structures of ribosomes were obtained at nanometer resolution from the spinach and liver samples.

冷冻电子断层扫描 (cryo-ET) 为原位研究大分子的完整结构提供了一种有前途的方法，但高质量冷冻切片的有效制备是一个瓶颈。尽管冷冻聚焦离子束 (cryo-FIB) 铣削已用于大而扁平的冷冻薄片制备，但其在组织标本中的应用仍然具有挑战性。在这里，我们报告了一个集成的工作流程，VHUT-cryo-FIB，用于有效地制备冷冻水合组织薄片，可以很容易地用于后续的冷冻 ET 研究。工作流程包括振动切片、高压冷冻、超薄切片机冷冻修整和冷冻-FIB 铣削。开发了两种通过侧入式低温支架或 FEI AutoGrid 装载低温薄片的策略。通过使用各种组织标本（包括大鼠骨骼肌、大鼠肝脏和菠菜叶标本）和原位验证了工作流程从菠菜和肝脏样本中以纳米分辨率获得核糖体的结构。