Related Experiment Video
Updated: Jun 15, 2026
09:06
Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography
Published on: November 20, 2021
Micromachining tools and correlative approaches for cellular cryo-electron tomography
Alexander Rigort1, Felix J B Bäuerlein, Andrew Leis
1Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Journal of Structural Biology
|February 25, 2010
Summary
Cryo-transmission electron microscopy (cryo-TEM) is limited by specimen thickness. Focused ion beam (FIB) milling and a novel cryo-planing method overcome this, enabling detailed cryo-electron tomography of cellular structures.
Area of Science:
- Structural Biology
- Microscopy Techniques
- Cell Biology
Background:
- Cryo-transmission electron microscopy (cryo-TEM) is crucial for visualizing cellular structures at high resolution.
- A key limitation in cryo-TEM, especially for tomography, is the restricted specimen thickness, which is worsened by tilting.
- Conventional cryo-ultramicrotomy can introduce mechanical deformations and yields poor-quality sections for tomographic imaging.
Purpose of the Study:
- To address the specimen thickness limitation in cryo-TEM for cellular and tissue analysis.
- To develop and validate advanced methods for preparing frozen-hydrated specimens for cryo-electron tomography.
- To enable high-resolution imaging of specific cellular targets within larger cellular volumes.
Main Methods:
- Utilized correlative cryo-fluorescence microscopy for navigation and target localization within large cellular volumes.
- Employed focused ion beam (FIB) milling for precise thinning of frozen-hydrated specimens via heavy ion sputtering (gallium ions).
- Introduced a novel cryo-planing procedure to thin large areas of vitreous ice before subsequent imaging steps.
Main Results:
- Demonstrated successful direct access to selected cellular targets in frozen-hydrated specimens using FIB milling.
- Showcased the effectiveness of FIB milling in overcoming specimen thickness limitations for cryo-electron tomography.
- Validated cryo-planing as a viable method for preparing large areas of vitreous ice for advanced cryo-EM workflows.
Conclusions:
- Focused ion beam milling offers a mechanical deformation-free alternative for preparing specimens for cryo-electron tomography.
- The combination of cryo-fluorescence microscopy, FIB milling, and cryo-planing significantly enhances the feasibility of cryo-TEM on thicker specimens.
- These advancements facilitate detailed structural analysis of cellular targets within their native context using cryo-electron tomography.
Related Concept Videos
01:07Electron Microscope Tomography and Single-particle Reconstruction
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.
Electron Tomography
Electron tomography can be performed either in TEM or STEM (scanning transmission...
Electron Tomography
Electron tomography can be performed either in TEM or STEM (scanning transmission...
01:28Cryo-electron Microscopy
Conventional electron microscopy (EM) involves dehydration, fixation, and staining of biological samples, which distorts the native state of biological molecules and results in several artifacts. Also, the high-energy electron beam damages the sample and makes it difficult to obtain high-resolution images. These issues can be addressed using cryo-EM, which uses frozen samples and gentler electron beams. The technique was developed by Jacques Dubochet, Joachim Frank, and Richard Henderson, for...