Journal
JOURNAL OF STRUCTURAL BIOLOGY
Volume 213, Issue 3, Pages -Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.jsb.2021.107763
Keywords
Cryofocused ion beam; Cryo-electron tomography; High-pressure freezing; Vibratome; Ultramicrotome cryo-trimming
Funding
- National Natural Science Foundation of China [31830020, 31770919, 31725007, 31630087]
- Ministry of Science and Technology of China [2017YFA0504700]
- Chinese Academy of Sciences [XDB37040102]
- Beijing Municipal Science and Technology Commission [Z181100004218002]
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This study reports an integrated workflow, VHUT-cryo-FIB, for efficiently preparing frozen hydrated tissue lamella for cryo-electron tomography studies. The workflow was validated by obtaining in situ structures of ribosomes from spinach and liver samples.
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 pre -paring 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 spec-imens, and in situ structures of ribosomes were obtained at nanometer resolution from the spinach and liver samples.
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