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Electron Microscope Tomography and Single-particle Reconstruction

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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.
Electron Tomography
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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...

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The cytoskeletal architecture can be studied using different microscopic and biochemical techniques. Electron microscopy was instrumental in discovering the cytoskeletal architecture around the 1960s, which allowed obtaining structural information at a high-resolution level. However, the sample preparation procedure often limits this ability in biological samples. Several protocols have been developed over the years to optimize sample preparation. In one of the protocols known as rotary...

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Related Experiment Video

Updated: Jun 3, 2025

Electron Cryotomography of Bacterial Cells

Electron Cryotomography of Bacterial Cells

Published on: May 6, 2010

Structural biology inside multicellular specimens using electron cryotomography.

Ido Caspy¹, Zhexin Wang¹, Tanmay A M Bharat¹

¹Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge, UK.

Quarterly Reviews of Biophysics

|January 13, 2025

Summary

This summary is machine-generated.

Electron cryotomography (cryo-ET) allows visualizing cellular structures in 3D. This review details current cryo-ET methods for multicellular specimens, identifies challenges, and suggests future improvements for in situ structural biology.

Keywords:

FIB-SEM cryo-EM cryo-ET focused ion beam milling in situ imaging structural biology subtomogram averaging tissue ultrastructure vitrification volume imaging

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Last Updated: Jun 3, 2025

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Area of Science:

Structural Biology
Cell Biology
Biophysics

Background:

Electron cryomicroscopy (cryo-EM) has revolutionized structural biology.
Electron cryotomography (cryo-ET) enables visualization of native 3D organization in biological specimens.
Studying multicellular specimens with cryo-ET faces significant workflow challenges.

Purpose of the Study:

To review current in situ imaging techniques for multicellular specimens using cryo-ET.
To enumerate the limitations associated with existing cryo-ET workflows.
To identify future advancements needed for comprehensive in situ structural biology.

Main Methods:

Review of current techniques in sample preparation for cryo-ET.
Analysis of data collection strategies for multicellular specimens.
Examination of image analysis methods in cryo-ET.

Main Results:

Latest techniques for in situ imaging of multicellular specimens are outlined.
Limitations across sample preparation, data collection, and image analysis are detailed.
Success stories highlight progress in cryo-ET applications.

Conclusions:

Significant improvements are needed across the entire cryo-ET workflow for multicellular specimens.
Future hardware and software developments are crucial for advancing in situ structural biology.
Enhanced cryo-ET capabilities will drive new discoveries in molecular and cell biology.