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Related Concept Videos

Cryo-electron Microscopy01:28

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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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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: Aug 3, 2025

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CryoFIB milling large tissue samples for cryo-electron tomography.

Sihan Wang1,2,3,4,5, Heng Zhou1,2,3,4,5, Wei Chen6

  • 1Key Laboratory for Protein Sciences of Ministry of Education, School of Life Sciences, Tsinghua University, Beijing, 100084, China.

Scientific Reports
|April 11, 2023
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Summary

This study introduces a cryo-focused ion beam (cryoFIB) milling workflow for cryo-electron tomography (cryoET). It enables efficient isolation of molecular structures from large tissue samples for detailed analysis.

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Area of Science:

  • Structural biology
  • Biophysics
  • Electron microscopy

Background:

  • Cryo-electron tomography (cryoET) is vital for molecular structure determination in large organisms.
  • Technical hurdles in sample preparation limit cryoET's utility for extensive biological specimens.
  • Precise localization and isolation of target structures within large tissues remain challenging.

Purpose of the Study:

  • To develop and present an optimized sample thinning strategy and workflow for cryo-electron tomography (cryoET) of large tissue samples.
  • To provide a comprehensive solution for isolating regions of interest from millimeter-sized tissues down to nanometer-scale lamellae.
  • To enhance the efficiency and feasibility of cryo-electron tomography for large biological samples.

Main Methods:

  • A novel workflow integrating sample fixation, pre-sectioning, and cryo-focused ion beam (cryoFIB) milling.
  • A two-step milling strategy involving coarse and fine milling stages to create a furrow-ridge structure.
  • Integration of cellular secondary electron imaging (CSEI) for real-time localization of target structures during milling.
  • Application of a platinum (Pt) layer to mitigate beam-induced charging effects.

Main Results:

  • Demonstration of a complete workflow for preparing large tissue samples for cryoET, from millimeter-sized starting material to hundred-nanometer-thin lamellae.
  • Successful isolation of objects of interest with high efficiency and feasibility.
  • Validation of the two-step milling strategy and CSEI for improved cryoFIB milling outcomes.
  • Reduction in beam-induced charging issues through the furrow-ridge structure and Pt coating.

Conclusions:

  • The presented cryoFIB milling workflow offers a robust solution for preparing large tissue samples for high-resolution cryoET.
  • The integrated approach, including CSEI and a two-step milling strategy, significantly improves the efficiency and success rate of isolating molecular targets.
  • This method expands the applicability of cryoET to larger biological samples, facilitating deeper insights into molecular organization and function.