期刊
JOURNAL OF STRUCTURAL BIOLOGY
卷 210, 期 2, 页码 -出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.jsb.2020.107488
关键词
Cryo-FIB; Cryo-lamella; Automation; cryo-EM; In situ structural biology
资金
- Clive and Vera Ramaciotti Platform for Structural Cryo-Electron Microscopy (Monash University, Clayton)
- Australian Research Council Laureate Fellowship
- ARC Centre of Excellence in Advanced Molecular Imaging
- National Health and Medical Research Council of Australia
Cryo-transmission electron tomography (cryo-ET) in association with cryo-focused ion beam (cryo-FIB) milling enables structural biology studies to be performed directly within the cellular environment. Cryo-preserved cells are milled and a lamella with a typical thickness of 200-300 nm provides an electron transparent window suitable for cryo-ET imaging. Cryo-FIB milling is an effective method, but it is a tedious and time-consuming process, which typically results in similar to 10 lamellae per day. Here, we introduce an automated method to reproducibly prepare cryo-lamellae on a grid and reduce the amount of human supervision. We tested the routine on cryo-preserved Saccharomyces cerevisiae, mammalian 293 T cells, and lysozyme protein crystals. Here we demonstrate that our method allows an increased throughput, achieving a rate of 5 lamellae/hour without the need to supervise the FIB milling. We demonstrate that the quality of the lamellae is consistent throughout the preparation and their compatibility with cryo-ET analyses.
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