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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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Pure substances consist of only one type of matter. A pure substance can be an element or a compound. An element consists of only one type of atom, while a compound consists of two or more types of atoms held together by a chemical bond. Elements are classified as atomic or molecular based on the nature of their basic units.
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Updated: Jan 22, 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: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

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High-Throughput Protein Analysis Using Negative Stain Electron Microscopy and 2D Classification.

Christopher P Arthur1, Claudio Ciferri2

  • 1Department of Structural Biology, Cryo-EM Unit. 1 DNA Way, MS, Genentech, Inc., South San Francisco, CA, USA. arthur.christopher@gene.com.

Methods in Molecular Biology (Clifton, N.J.)
|July 4, 2019
PubMed
Summary
This summary is machine-generated.

Negative staining electron microscopy quickly assesses protein properties like architecture and flexibility, complementing high-throughput methods. This technique aids in understanding protein complexes and antibody-antigen interactions.

Keywords:
2D classificationEpitope mappingNegative stain EMProtein expressionSingle particle analysisTransmission electron microscopy

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Microscopy

Background:

  • High-throughput protein expression and purification are vital for selecting protein constructs.
  • Current methods often fail to provide insights into crucial biochemical properties like domain architecture, homogeneity, and flexibility.
  • There is a need for rapid assessment of these properties to complement existing high-throughput techniques.

Purpose of the Study:

  • To describe a protocol for negative staining electron microscopy (EM) for protein analysis.
  • To demonstrate the application of negative staining EM coupled with single particle analysis for evaluating protein complexes.
  • To highlight the utility of this method for determining antibody binding sites on antigens.

Main Methods:

  • Development and description of a protocol for negative stain sample preparation.
  • Imaging of protein samples using electron microscopy.
  • Two-dimensional (2D) data analysis of negative stain electron microscopy images.
  • Application to single particle analysis for structural and conformational insights.

Main Results:

  • The protocol is applicable to a wide variety of protein complexes.
  • Negative staining EM provides rapid evaluation of protein domain architecture, homogeneity, and flexibility.
  • The method successfully informed on the architecture and conformational state of protein samples.
  • Specific application demonstrated successful determination of antibody binding sites on target antigens.

Conclusions:

  • Negative staining electron microscopy is a valuable tool for rapid biochemical characterization of proteins and complexes.
  • Coupling negative staining EM with single particle analysis offers detailed structural information.
  • This approach significantly enhances the ability to study large molecules and molecular interactions, such as antibody-antigen binding.