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Related Concept Videos

Overview of Electron Microscopy01:25

Overview of Electron Microscopy

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The wavelengths of visible light ultimately limit the maximum theoretical resolution of images created by light microscopes. Most light microscopes can only magnify 1000X, and a few can magnify up to 1500X. Electrons, like electromagnetic radiation, can behave like waves, but with wavelengths of 0.005 nm, they produce significantly greater resolution up to 0.05 nm as compared to 500 nm for visible light. An electron microscope (EM) can create a sharp image that is magnified up to 2,000,000X.
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A scanning electron microscope (SEM) is used to study the surface features of a sample by using an electron beam that scans the sample surface in a two-dimensional manner. Typically, areas between ~1 centimeter to 5 micrometers in width can be imaged. SEM can be used to image bacteria, viruses, tissues as well as larger samples like insects. Conventional SEM gives a magnification ranging from 20X to 30,000X and spatial resolution of 50 to 100 nanometers.
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Transmission Electron Microscopy01:15

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In 1931, physicist Ernst Ruska—building on the idea that magnetic fields can direct an electron beam just as lenses can direct a beam of light in an optical microscope—developed the first prototype of the electron microscope. This development led to the development of the field of electron microscopy. In the transmission electron microscope (TEM), electrons are produced by a hot tungsten element and accelerated by a potential difference in an electron gun, which gives them up to 400...
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Immunogold Electron Microscopy01:20

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Immunoelectron microscopy utilizes immunogold labeling of endogenous proteins with specific antibodies to detect and localize these proteins in cells and tissues. The procedure provides insights into the distribution and quantification of protein under different stimulation conditions offering clues about their functions. Conjugating highly electron-dense gold particles with primary or secondary antibodies allow antigen detection on and within cells, with high resolution and specificity.
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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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Preparation of Samples for Electron Microscopy01:20

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To be visualized by an electron microscope, either transmission or scanning, biological samples need to be fixed (stabilized) so the electron beam does not destroy them and dried thoroughly (desiccated/dehydrated) so the vacuum does not affect them. Fixation needs to be done as quickly as possible because the sample properties will start changing as soon as it is removed from its natural environment. For example, in a tissue sample, the oxygen levels begin decreasing, causing an altered...
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Related Experiment Video

Updated: Jan 23, 2026

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy
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Optimized Negative-Staining Protocol for Lipid-Protein Interactions Investigated by Electron Microscopy.

Jianfang Liu1, Hao Wu1,2, Changyu Huang1

  • 1Molecular Foundry, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.

Methods in Molecular Biology (Clifton, N.J.)
|June 21, 2019
PubMed
Summary

Researchers optimized negative-staining electron microscopy (NS-EM) to visualize lipid-binding proteins. This new method overcomes artifacts, providing near native-state images for better structural analysis of protein-lipid interactions.

Keywords:
Electron microscopyLipoprotein morphologyLipoprotein structureNegative-staining electron microscopyOptimized negative-staining protocol

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Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy
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Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

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Area of Science:

  • Structural Biology
  • Biophysics
  • Biochemistry

Background:

  • Lipid-protein interactions are crucial for biological processes.
  • Determining protein structures at lipid-binding stages is vital for understanding protein function.
  • Traditional methods like X-ray crystallography struggle with the heterogeneity of protein-lipid complexes.

Purpose of the Study:

  • To develop an optimized negative-staining electron microscopy (NS-EM) protocol for visualizing lipid-protein complexes.
  • To overcome artifacts commonly observed with lipid-related proteins using standard NS-EM.
  • To enable high-quality structural determination of proteins in their native lipid-binding state.

Main Methods:

  • Refinement of existing NS-EM protocols.
  • Validation of the optimized protocol by comparing images with cryo-electron microscopy (cryo-EM) data.
  • Application of the optimized protocol for single-particle analysis and electron tomography (e.g., IPET).

Main Results:

  • The optimized NS-EM protocol effectively minimizes artifacts like rouleaux formation in lipoproteins.
  • It produces high-contrast images of proteins in their near native-state lipid-binding conformation.
  • The protocol facilitates higher-quality three-dimensional (3D) reconstructions.

Conclusions:

  • The optimized NS-EM protocol is a valuable and practical tool for studying protein structures at the lipid-binding stage.
  • This method enhances the understanding of protein-lipid interactions and their functional implications.
  • It offers a promising approach for structural determination where traditional methods fall short.