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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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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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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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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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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 of Bone.

Alan Boyde1

  • 1Dental Physical Sciences, Biophysics Section, Oral Growth and Development, Dental Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK. a.boyde@qmul.ac.uk.

Methods in Molecular Biology (Clifton, N.J.)
|February 8, 2019
PubMed
Summary
This summary is machine-generated.

This chapter details scanning electron microscopy (SEM) sample preparation for bone and bone cells. It highlights backscattered electron (BSE) imaging and maceration techniques for detailed 3D bone structure analysis.

Keywords:
MineralizationMorphologyMouse geneticsOsteoarthritisOsteoporosis

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Area of Science:

  • Biomaterials Science
  • Microscopy Techniques
  • Skeletal Biology

Background:

  • Scanning electron microscopy (SEM) is crucial for analyzing bone and bone cell structures.
  • Effective sample preparation is essential for high-quality SEM imaging, particularly for complex biological tissues like bone.
  • Previous methods faced challenges with sample integrity, charging artifacts, and detailed visualization of cellular and matrix components.

Purpose of the Study:

  • To provide comprehensive methods for preparing bone and bone cell samples for various SEM imaging modes.
  • To detail techniques for achieving high-resolution 3D imaging of bone microarchitecture using backscattered electron (BSE) SEM.
  • To offer guidance on sample handling, embedding, maceration, and artifact mitigation for SEM analysis of bone.

Main Methods:

  • Sample preparation techniques including fixation, drying, and metallic conductive coating for bone cells.
  • Maceration protocols using enzymes and chemicals (pronase, hypochlorite, hydrogen peroxide, hydroxides) to remove cellular and unmineralized matrix components.
  • Resin embedding (including PMMA) for SEM, spatial cast creation, and preparation for correlated imaging (confocal, microradiography, microtomography).

Main Results:

  • Backscattered electron (BSE) imaging identified as the most valuable SEM mode for bone analysis.
  • Detailed methods for 3D BSE SEM imaging of bone samples, including resin embedding recommendations.
  • Successful visualization of bone undersides, spatial casts, and mineralizing front labels using cathodoluminescence (CL) mode SEM.

Conclusions:

  • Standardized and advanced SEM preparation methods enable detailed structural analysis of bone and bone cells.
  • 3D BSE SEM imaging, combined with appropriate sample preparation, offers significant insights into bone microarchitecture.
  • Control of SEM vacuum pressure and proper sample embedding are key to overcoming common imaging challenges like charging artifacts.