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Cryo-electron Microscopy

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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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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

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.
Fundamental Principles
Accelerated...
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Phase Contrast and Differential Interference Contrast Microscopy01:26

Phase Contrast and Differential Interference Contrast Microscopy

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Phase-Contrast Microscopes
In-phase-contrast microscopes, interference between light directly passing through a cell and light refracted by cellular components is used to create high-contrast, high-resolution images without staining. It is the oldest and simplest type of microscope that creates an image by altering the wavelengths of light rays passing through the specimen. Altered wavelength paths are created using an annular stop in the condenser. The annular stop produces a hollow cone of...
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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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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.
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Micrograph contrast in low-voltage SEM and cryo-SEM.

Lucy Liberman1, Olga Kleinerman2, Irina Davidovich1

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Summary

Modern scanning electron microscopes (SEM) offer high-resolution imaging of various materials. Optimizing acceleration voltage and detector choice enhances contrast, crucial for analyzing nanomaterials and biological samples.

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Cryo-SEMElectron beam accelerating voltageLow-voltage SEMPicture contrast

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Area of Science:

  • Materials Science
  • Nanotechnology
  • Microscopy

Background:

  • Modern high-resolution scanning electron microscopes (SEM) with field emission guns (FEGs) enable detailed study of conductive and insulating materials without coating.
  • Advancements in electron sources, optics, vacuum, and detectors enhance SEM's capability as a powerful characterization tool for nanomaterials.
  • Exploiting slight specimen charging can improve contrast between different phases, minimizing imaging artifacts for diverse materials.

Purpose of the Study:

  • To investigate the impact of acceleration voltage and detector selection on contrast formation in SEM imaging.
  • To optimize imaging parameters for materials with inherently low contrast, such as carbon- and oxygen-based specimens.
  • To demonstrate the utility of cryogenic SEM (cryo-SEM) for studying specimens in their native states, including emulsions and carbon nanotubes.

Main Methods:

  • Utilized high-resolution SEM (HR-SEM) at room temperature and cryogenic SEM (cryo-SEM).
  • Examined carbon nanotubes (CNTs) dispersed in water, dissolved in superacid, and as films on glass.
  • Varied acceleration voltage and employed different detectors to observe changes in micrograph contrast.

Main Results:

  • Demonstrated how micrograph contrast is affected by different detectors and acceleration voltages.
  • Showcased the ability to achieve optimal contrast between domains of varying composition through judicious SEM parameter selection.
  • Successfully imaged emulsions and CNTs in different states using cryo-SEM and HR-SEM.

Conclusions:

  • SEM operational parameters, specifically acceleration voltage and detector choice, are critical for achieving high contrast in imaging low-contrast materials.
  • Optimized SEM imaging is essential for understanding structure-property relationships in nanomaterials and biological systems.
  • The study provides a framework for selecting appropriate SEM settings to maximize information retrieval from diverse specimens.