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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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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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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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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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Ultrastructural Localization of Endogenous LC3 by On-Section Correlative Light-Electron Microscopy
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High-throughput ultrastructure screening using electron microscopy and fluorescent barcoding.

Yury S Bykov1,2, Nir Cohen3, Natalia Gabrielli1

  • 1Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.

The Journal of Cell Biology
|July 11, 2019
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Summary
This summary is machine-generated.

High-throughput electron microscopy (EM) now enables screening of multiple cell types from single samples. This MultiCLEM method uses fluorescent barcodes and correlative light and EM to analyze cellular ultrastructure at scale.

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Area of Science:

  • Cell Biology
  • Microscopy
  • Biotechnology

Background:

  • High-throughput genetic screens using fluorescent microscopy yield cell biology insights but lack ultrastructural resolution.
  • Electron microscopy (EM) offers detailed ultrastructure but is limited by low throughput and time-consuming sample preparation.

Purpose of the Study:

  • To develop a robust method for high-throughput screening using EM.
  • To enable the analysis of multiple cell populations within a single EM sample preparation.

Main Methods:

  • Developed MultiCLEM, a method combining fluorescent barcodes for cell identification with correlative light and EM (CLEM).
  • Utilized a software workflow for automated correlation, segmentation, and image analysis.
  • Demonstrated the method using 15 different yeast variants.

Main Results:

  • MultiCLEM successfully extracts and analyzes multiple cell populations from each EM sample.
  • The method allows for screening of cellular ultrastructure at a throughput compatible with genetic screens.
  • Successfully applied to distinguish and analyze different yeast variants.

Conclusions:

  • MultiCLEM significantly enhances the throughput of EM, transforming it into a powerful screening technique.
  • The methodology is versatile, scalable, and applicable beyond yeast research.
  • Enables new avenues for cell biological discovery by integrating ultrastructural analysis into screening paradigms.