Perton Elise G, Boltje Daan B, Jakobi Arjen J, Hoogenboom Jacob P
Department of Imaging Physics, Delft University of Technology, Delft, The Netherlands.
Kavli Institute of Nanoscience, Delft University of Technology, Delft, The Netherlands.
Bio Protoc. 2025 Jul 20;15(14):e5390. doi: 10.21769/BioProtoc.5390.
Cryo-electron tomography (cryo-ET) is the main technique to image the structure of biological macromolecules inside their cellular environment. The samples for cryo-ET must be thinner than 200 nm, which is not compatible with micron-sized cells. A focused ion beam (FIB), in conjunction with a scanning electron microscope (SEM) to navigate the sample, can be used to ablate material from vitrified cells such that a thin lamella remains. However, the preparation of lamellae with a FIB-SEM is blind to the location of specific cellular structures and biomolecules. Furthermore, the thickness and uniformity of lamella, while crucial for high-quality tomograms, cannot be established accurately with the FIB-SEM. These limitations strongly affect the success rate for cryo-ET on FIB-milled lamellae and thereby the total throughput of the workflow. To mitigate these problems, a coincident light, electron, and ion beam cryo-microscope was developed by retrofitting a fluorescence microscope, cryogenic microcooler, and piezo stage on a FIB-SEM. The fluorescence of molecules of interest can be monitored in real time while milling to ensure the final lamella contains the structure of interest. In addition, reflected light microscopy can be used for thickness and quality control of the lamella. In this protocol, we will describe how the coincident microscope can be used to prepare lamellae from vitrified cells. Key features • Step-by-step protocol for fluorescence-guided FIB-milling with a coincident three-beam cryogenic microscope as described in [1]. • Details about sample loading and unloading, as well as the lamella milling workflow with graphical explanations. • Quality control of lamella, including thickness, uniformity, and ice contamination.
冷冻电子断层扫描（cryo-ET）是用于对细胞环境中生物大分子结构进行成像的主要技术。cryo-ET的样本必须薄于200纳米，这与微米级大小的细胞不兼容。聚焦离子束（FIB）与扫描电子显微镜（SEM）结合用于引导样本，可以用来从玻璃化细胞中消融材料，从而留下一个薄片。然而，使用FIB-SEM制备薄片对特定细胞结构和生物分子的位置是盲目的。此外，薄片的厚度和均匀性对于高质量的断层扫描至关重要，但使用FIB-SEM无法准确确定。这些限制严重影响了在FIB铣削薄片上进行cryo-ET的成功率，进而影响了整个工作流程的总通量。为了缓解这些问题，通过在FIB-SEM上加装荧光显微镜、低温微型冷却器和压电平台，开发了一种同时具备光、电子和离子束的冷冻显微镜。在铣削过程中可以实时监测目标分子的荧光，以确保最终的薄片包含目标结构。此外，反射光显微镜可用于薄片的厚度和质量控制。在本方案中，我们将描述如何使用这种同时具备三种光束的显微镜从玻璃化细胞中制备薄片。关键特性 • 如[1]中所述，使用同时具备三种光束的低温显微镜进行荧光引导FIB铣削的分步方案。 • 关于样本加载和卸载的详细信息，以及带有图形解释的薄片铣削工作流程。 • 薄片的质量控制，包括厚度、均匀性和冰污染。
