CryoFIB milling large tissue samples for cryo-electron tomography

Cryo-electron tomography (cryoET) is a powerful tool for exploring the molecular structure of large organisms. However, technical challenges still limit cryoET applications on large samples. In particular, localization and cutting out objects of interest from a large tissue sample are still difficul...

Full description

Saved in:
Bibliographic Details
Published inScientific reports Vol. 13; no. 1; pp. 5879 - 12
Main Authors Wang, Sihan, Zhou, Heng, Chen, Wei, Jiang, Yifeng, Yan, Xuzhen, You, Hong, Li, Xueming
Format Journal Article
LanguageEnglish
Published London Nature Publishing Group UK 11.04.2023
Nature Publishing Group
Nature Portfolio
Subjects
631/1647/2258/1258/1260
631/1647/328/1259
Cell Membrane
Cryoelectron Microscopy - methods
Electron Microscope Tomography - methods
Electrons
Humanities and Social Sciences
Ions - chemistry
Lamellae
Localization
multidisciplinary
Science
Science (multidisciplinary)
Sectioning
Tomography
Workflow
Online AccessGet full text

Cover

More Information
Summary:Cryo-electron tomography (cryoET) is a powerful tool for exploring the molecular structure of large organisms. However, technical challenges still limit cryoET applications on large samples. In particular, localization and cutting out objects of interest from a large tissue sample are still difficult steps. In this study, we report a sample thinning strategy and workflow for tissue samples based on cryo-focused ion beam (cryoFIB) milling. This workflow provides a full solution for isolating objects of interest by starting from a millimeter-sized tissue sample and ending with hundred-nanometer-thin lamellae. The workflow involves sample fixation, pre-sectioning, a two-step milling strategy, and localization of the object of interest using cellular secondary electron imaging (CSEI). The milling strategy consists of two steps, a coarse milling step to improve the milling efficiency, followed by a fine milling step. The two-step milling creates a furrow–ridge structure with an additional conductive Pt layer to reduce the beam-induced charging issue. CSEI is highlighted in the workflow, which provides on-the-fly localization during cryoFIB milling. Tests of the complete workflow were conducted to demonstrate the high efficiency and high feasibility of the proposed method.
Bibliography:ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-023-32716-z