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Scanning Electron Microscopy

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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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Overview of Electron Microscopy01:25

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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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The early pioneers of microscopy opened a window into the invisible world of microorganisms. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th century saw the development of microscopes that leveraged nonvisible light, such as fluorescence microscopy that uses an ultraviolet light source and electron microscopy that uses short-wavelength electron beams. These advances significantly improved magnification, image resolution, and contrast. By comparison, the...
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Electron Microscope Tomography and Single-particle Reconstruction01:07

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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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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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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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Scanning Electron Microscopy.

Elizabeth R Fischer1, Bryan T Hansen1, Vinod Nair1

  • 1Electron Microscopy Unit, Research Technologies Branch, Rocky Mountain Laboratories, National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana.

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Summary
This summary is machine-generated.

This guide details essential scanning electron microscopy (SEM) techniques for biological specimens. Learn optimal preparation and imaging methods to minimize artifacts and achieve high-resolution topographical visualization.

Keywords:
EM specimen preparationcritical point dryingcryo‐SEM quantum dotsimmune‐labelingmicrowave‐processingscanning electron microscopyspecimen fracturingsputter coating

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Area of Science:

  • Microscopy
  • Materials Science
  • Biology

Background:

  • Scanning electron microscopy (SEM) is crucial for visualizing specimen topography.
  • Specimen preparation is critical to minimize artifacts and preserve structural integrity.
  • Standard SEM operation requires specimens to be compatible with high-vacuum environments.

Purpose of the Study:

  • To provide fundamental techniques and tips for routine biological specimen preparation for SEM.
  • To guide users in preserving labile or fragile structures.
  • To detail immune-labeling strategies and microscope imaging parameters for optimal SEM examination.

Main Methods:

  • Chemical fixation and dehydration protocols to reduce structural artifacts and shrinkage.
  • Substrate selection and coating methods to mitigate charging artifacts.
  • Critical point drying and alternative methods for specimen stabilization.
  • Immune-labeling strategies for enhanced structural visualization.
  • Mechanical and cryo-fracturing techniques for exposing internal structures.
  • Sputter coating for improved conductivity.

Main Results:

  • Demonstrates methods to minimize chemical processing artifacts during SEM specimen preparation.
  • Outlines dehydration techniques to prevent shrinkage for high-vacuum compatibility.
  • Presents strategies for reducing charging artifacts through substrate choice and coating.
  • Details protocols for preserving delicate biological structures.
  • Explains immune-labeling and fracturing techniques for detailed structural analysis.

Conclusions:

  • Mastering SEM specimen preparation is key to accurate topographical visualization of biological samples.
  • Careful selection of fixation, dehydration, mounting, and imaging parameters minimizes artifacts.
  • This article serves as a comprehensive resource for routine SEM examination of diverse biological specimens.