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Related Experiment Video

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Preparing Lamellae from Vitreous Biological Samples Using a Dual-Beam Scanning Electron Microscope for Cryo-Electron Tomography
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Mind the gap: Micro-expansion joints drastically decrease the bending of FIB-milled cryo-lamellae.

Georg Wolff1, Ronald W A L Limpens1, Shawn Zheng2

  • 1Department of Cell and Chemical Biology, Section Electron Microscopy, Leiden University Medical Center, Leiden 2333 ZC, The Netherlands.

Journal of Structural Biology
|September 20, 2019
PubMed
Summary
This summary is machine-generated.

Cryo-focused ion beam (FIB) milling now includes micro-expansion joints to prevent cryo-lamella bending. This simple adaptation improves throughput for in situ structural biology studies.

Keywords:
Cellular cryotomographyCryo-FIB-millingCryo-electron tomographyCryo-lamellaFrozen-hydrated cellsLamella bending

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Area of Science:

  • Structural Biology
  • Cryo-electron Microscopy
  • Biophysics

Background:

  • Cryo-focused ion beam (FIB) milling is crucial for preparing thin cryo-lamellae from biological samples for in situ cryo-electron tomography.
  • Lamella bending during cryo-FIB milling, often caused by EM grid support film crinkling at cryogenic temperatures, is a major limitation, reducing throughput and sample integrity.
  • This bending can lead to lamella breakage, hindering high-resolution structural studies of cellular components in their native environment.

Purpose of the Study:

  • To investigate the cause of cryo-lamella bending during cryo-FIB milling.
  • To develop and validate a novel method to mitigate cryo-lamella bending.
  • To enhance the throughput and reliability of the cryo-lamella workflow for in situ structural biology.

Main Methods:

  • Milling of "micro-expansion joints" adjacent to cryo-lamellae during the cryo-FIB process.
  • Comparative analysis of lamella bending in samples with and without micro-expansion joints.
  • Acquisition and analysis of high-quality Volta phase plate tomograms from prepared cryo-lamellae.

Main Results:

  • Micro-expansion joints significantly reduced lamella bending in eukaryotic cells.
  • The presence of micro-expansion joints did not introduce new instabilities into the cryo-lamella workflow.
  • High-resolution Volta phase plate tomograms were successfully obtained, showcasing cellular macromolecules in their native context.

Conclusions:

  • Micro-expansion joints serve as effective physical buffers, absorbing tensions that cause lamella bending.
  • This straightforward modification minimally increases workflow complexity while substantially improving cryo-lamella integrity.
  • The micro-expansion joint technique offers a practical solution to increase the throughput of in situ structural biology research.