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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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Scanning Electron Microscopy01:07

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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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Preparation of Samples for Electron Microscopy01:20

Preparation of Samples for Electron Microscopy

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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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Confocal Fluorescence Microscopy01:16

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Confocal microscopy is an advanced microscopic technique. The prime advantage of the confocal microscope over other microscopy techniques is its ability to block the out-of-focus light from the illuminated samples using pinholes. It is widely used with fluorescence optics to obtain high-resolution, sharp contrast images. Unlike optical microscopes, confocal microscopes use a focused beam of light laser to scan the entire sample surface at different z-planes. These microscopes are, therefore,...
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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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Overview of Microscopy Techniques01:22

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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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Author Spotlight: Advancements in Correlative Light and Electron Microscopy with Fluorescent Protein Preservation
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Correlative light-electron microscopy in liquid using an inverted SEM (ASEM).

Chikara Sato1, Takaaki Kinoshita2, Nassirhadjy Memtily3

  • 1National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan; University of Tsukuba, Tsukuba, Japan.

Methods in Cell Biology
|May 23, 2017
PubMed
Summary

Atmospheric scanning electron microscopy (ASEM) enables high-resolution imaging of wet biological samples by combining inverted scanning electron microscopy with optical microscopy. This technique visualizes cellular structures and phenomena in detail, advancing mesoscopic biological research.

Keywords:
Atmospheric scanning electron microscopy (ASEM)Bacterial floraBiofilmDiabetes mellitusIntra operative cancer diagnosisSilicon nitride film

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Area of Science:

  • Life Sciences
  • Microscopy
  • Cell Biology

Background:

  • Traditional electron microscopy requires samples to be in a vacuum, limiting the observation of hydrated biological specimens.
  • Observing dynamic biological processes in their native, hydrated state is crucial for understanding cellular function.

Purpose of the Study:

  • To introduce and validate the atmospheric scanning electron microscope (ASEM) for high-resolution imaging of wet biological samples.
  • To demonstrate the capability of ASEM in visualizing cellular structures and biological phenomena in real-time.

Main Methods:

  • Utilizing an inverted scanning electron microscope (SEM) combined with simultaneous top-down optical microscopy.
  • Employing a specialized sample holder with a silicon nitride film window for wet sample observation.
  • Applying aldehyde fixation, radical scavenger immersion, and minimal electron dose for SEM imaging.
  • Integrating correlative light and electron microscopy (CLEM) techniques.

Main Results:

  • Successfully visualized neural networking, axonal segmentation, proplatelet formation, phagocytosis, and Fas expression in embryonic stem cells.
  • High-resolution imaging of islet of Langerhans, blood microvessels, neuronal endplates, and stomach bacterial flora.
  • Detailed visualization of bacterial biofilms and mycoplasma structures, including the "leg complex" using CLEM.
  • Demonstrated the ability to locate regions of interest with optical microscopy before high-resolution SEM examination.

Conclusions:

  • ASEM provides a powerful platform for observing hydrated biological samples at high resolution.
  • The technique facilitates the study of various cellular processes and tissue structures in a near-native environment.
  • ASEM correlative microscopy holds significant promise for future research in mesoscopic biological phenomena.