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Related Concept Videos

Electron Microscope Tomography and Single-particle Reconstruction01:07

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Transmission electron microscopy (TEM) can be used to determine the 3D structure of biological samples with the help of techniques such as electron microscope tomography and single-particle reconstruction. While single-particle reconstruction can examine macromolecules and macromolecular complexes in vitro conditions only, tomography permits the study of cell components or small cells in vivo.
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Related Experiment Video

Updated: Dec 26, 2025

Sample Preparation by 3D-Correlative Focused Ion Beam Milling for High-Resolution Cryo-Electron Tomography
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Fully automated, sequential focused ion beam milling for cryo-electron tomography.

Tobias Zachs1, Andreas Schertel2, João Medeiros1

  • 1Institute of Molecular Biology and Biophysics, Eidgenössische Technische Hochschule Zürich, Zürich, Switzerland.

Elife
|March 10, 2020
PubMed
Summary

Automated cryo-focused ion beam (cryoFIB) milling streamlines sample preparation for cryo-electron tomography (cryoET). This innovation enables high-quality cellular imaging and structural analysis, making previously challenging projects feasible.

Keywords:
S. cerevisiaedual beam instrumentin situ imaginglamellamolecular biophysicssample preparationsample thinningstructural biologytomography

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Area of Science:

  • Structural Biology
  • Cell Biology
  • Microscopy

Background:

  • Cryo-electron tomography (cryoET) enables in-situ imaging of cellular structures.
  • Cryo-electron tomography (cryoET) is limited by sample thickness.
  • Cryo-focused ion beam (cryoFIB) milling thins samples for cryoET but is manual and slow.

Purpose of the Study:

  • To develop a fully automated method for sequential cryo-focused ion beam (cryoFIB) milling.
  • To improve the throughput and reduce hands-on time for cryoET sample preparation.
  • To enable high-quality cryoET imaging and analysis of challenging biological samples.

Main Methods:

  • Developed a novel automated sequential cryoFIB milling protocol.
  • Included automated rough milling and polishing steps.
  • Applied the method to both eukaryotic and bacterial model organisms.

Main Results:

  • Successfully prepared high-quality lamellae suitable for cryoET.
  • Demonstrated reproducible results across different model organisms.
  • Achieved significant reduction in sample preparation time and user intervention.

Conclusions:

  • The automated cryoFIB method enhances cryoET sample preparation efficiency.
  • This technique expands the scope of cryoET applications in structural and cell biology.
  • Automated lamellar preparation will facilitate previously inaccessible biological investigations.