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TitleVHUT-cryo-FIB, a method to fabricate frozen hydrated lamellae from tissue specimens for in situ cryo-electron tomography.
Journal, issue, pagesJ Struct Biol, Vol. 213, Issue 3, Page 107763, Year 2021
Publish dateJun 24, 2021
AuthorsJianguo Zhang / Danyang Zhang / Lei Sun / Gang Ji / Xiaojun Huang / Tongxin Niu / Jiashu Xu / Chengying Ma / Yun Zhu / Ning Gao / Wei Xu / Fei Sun /
PubMed AbstractCryo-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 ...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.
External linksJ Struct Biol / PubMed:34174447
MethodsEM (tomography) / EM (subtomogram averaging)
Resolution34.0 Å
Structure data

EMDB-0732:
Cryo electron tomogram of cryo-lamella of rat skeleton muscle
Method: EM (tomography)

EMDB-0733:
Cryo electron tomogram of cryo-lamella of spinach leaf
Method: EM (tomography)

EMDB-0734:
Spinach 80S ribosome
Method: EM (subtomogram averaging) / Resolution: 34.0 Å

Source
  • Mus musculus (house mouse)
  • Spinacia oleracea (spinach)
  • Spinacia olerac (spinach)

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