Division of Nanoscopy, Maastricht Multimodal Molecular Imaging Institute, Maastricht University, the Netherlands.
Division of Nanoscopy, Maastricht Multimodal Molecular Imaging Institute, Maastricht University, the Netherlands.
J Struct Biol. 2021 Mar;213(1):107698. doi: 10.1016/j.jsb.2021.107698. Epub 2021 Feb 2.
Cryo-electron tomography (CET) on cryo-focused ion beam (FIB)-milled lamellae is becoming a powerful technique for determining the structure of macromolecular complexes in their native cellular environment. Prior to tomogram reconstruction, CET tilt-series recorded on FIB lamellae need to be aligned. Traditionally, CET tilt-series alignment is performed with 5-20 nm gold fiducials, but it has thus far proven difficult to apply this to FIB lamellae of eukaryotic cells. In here, we describe a simple method to allow uptake of bovine serum albumin (BSA)-gold fiducials into mammalian cells via endocytosis, which can subsequently be used as fiducials for tilt-series alignment of cryo-FIB lamellae. We compare the alignment of tilt-series with BSA-gold fiducials to fiducial-less patch-tracking, and find better alignment results with BSA-gold. This technique can contribute to understand cells at a structural and ultrastructural level with both cryo- and room-temperature electron tomography. Furthermore, fluorescently labeled BSA-gold has the potential to be used as fiducials for correlative light and electron microscopy studies.
冷冻聚焦离子束（FIB）铣削的薄片的冷冻电子断层扫描（CET）正在成为一种强大的技术，可用于确定其天然细胞环境中大分子复合物的结构。在进行断层重建之前，需要对 FIB 薄片上记录的 CET 倾斜系列进行对齐。传统上，使用 5-20nm 金标记来进行 CET 倾斜系列的对齐，但迄今为止，很难将其应用于真核细胞的 FIB 薄片。在这里，我们描述了一种简单的方法，可通过胞吞作用将牛血清白蛋白（BSA）-金标记物摄取到哺乳动物细胞中，随后可以将其用作冷冻 FIB 薄片倾斜系列对齐的标记物。我们将带有 BSA-金标记物的倾斜系列的对齐与无标记物的斑块跟踪进行了比较，并发现 BSA-金的对齐结果更好。该技术可用于在冷冻和室温电子断层扫描中以结构和超微结构水平来研究细胞。此外，荧光标记的 BSA-金有可能被用作相关的光和电子显微镜研究的标记物。
