He Jie, Hsieh Chyongere, Wu Yongping, Schmelzer Thomas, Wang Pan, Lin Ying, Marko Michael, Sui Haixin
Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, NY 12201, USA.
Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, NY 12201, USA; Zhejiang Agriculture and Forestry University, Zhejiang Province, PR China.
J Struct Biol. 2017 Aug;199(2):114-119. doi: 10.1016/j.jsb.2017.05.011. Epub 2017 May 27.
Cryo-electron tomography (cryo-ET) is a well-established technique for studying 3D structural details of subcellular macromolecular complexes and organelles in their nearly native context in the cell. A primary limitation of the application of cryo-ET is the accessible specimen thickness, which is less than the diameters of almost all eukaryotic cells. It has been shown that focused ion beam (FIB) milling can be used to prepare thin, distortion-free lamellae of frozen biological material for high-resolution cryo-ET. Commercial cryosystems are available for cryo-FIB specimen preparation, however re-engineering and additional fixtures are often essential for reliable results with a particular cryo-FIB and cryo-transmission electron microscope (cryo-TEM). Here, we describe our optimized protocol and modified instrumentation for cryo-FIB milling to produce thin lamellae and subsequent damage-free cryotransfer of the lamellae into our cartridge-type cryo-TEM.
冷冻电子断层扫描（cryo-ET）是一种成熟的技术，用于在细胞内接近天然状态下研究亚细胞大分子复合物和细胞器的三维结构细节。cryo-ET应用的一个主要限制是可处理的标本厚度，它小于几乎所有真核细胞的直径。研究表明，聚焦离子束（FIB）铣削可用于制备冷冻生物材料的薄且无变形的薄片，用于高分辨率cryo-ET。有商业化的冷冻系统可用于cryo-FIB标本制备，然而，对于特定的cryo-FIB和冷冻透射电子显微镜（cryo-TEM），重新设计和额外的固定装置通常对于获得可靠结果至关重要。在这里，我们描述了我们优化的cryo-FIB铣削方案和改进的仪器，以生产薄片，并随后将薄片无损冷冻转移到我们的盒式cryo-TEM中。
