VHUT-cryo-FIB, a method to fabricate frozen hydrated lamellae from tissue specimens for in situ cryo-electron tomography

Jianguo Zhang; Danyang Zhang; Lei Sun; Gang Ji; Xiaojun Huang; Tongxin Niu; Jiashu Xu; Chengying Ma; Yun Zhu; Ning Gao; Wei Xu; Fei Sun

doi:10.1016/j.jsb.2021.107763

VHUT-cryo-FIB, a method to fabricate frozen hydrated lamellae from tissue specimens for in situ cryo-electron tomography

J Struct Biol. 2021 Sep;213(3):107763. doi: 10.1016/j.jsb.2021.107763. Epub 2021 Jun 24.

Authors

Jianguo Zhang¹, Danyang Zhang², Lei Sun³, Gang Ji⁴, Xiaojun Huang³, Tongxin Niu¹, Jiashu Xu⁵, Chengying Ma⁶, Yun Zhu⁵, Ning Gao⁶, Wei Xu¹, Fei Sun⁷

Affiliations

¹ National Key Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
² National Key Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing, China; Physical Science Laboratory, Huairou National Comprehensive Science Center, No. 5 Yanqi East Second Street, Beijing 101400, China.
³ Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
⁴ University of Chinese Academy of Sciences, Beijing, China; Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
⁵ National Key Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing, China.
⁶ State Key Laboratory of Membrane Biology, Peking-Tsinghua Center for Life Sciences, School of Life Sciences, Peking University, Beijing 100871, China.
⁷ National Key Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing, China; Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Physical Science Laboratory, Huairou National Comprehensive Science Center, No. 5 Yanqi East Second Street, Beijing 101400, China. Electronic address: feisun@ibp.ac.cn.

PMID: 34174447
DOI: 10.1016/j.jsb.2021.107763

Abstract

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.

Keywords: Cryo-electron tomography; Cryofocused ion beam; High-pressure freezing; Ultramicrotome cryo-trimming; Vibratome.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cryoelectron Microscopy / methods
Electron Microscope Tomography* / methods
Freezing
Ions
Macromolecular Substances

Substances

Ions
Macromolecular Substances