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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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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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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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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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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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Software tools for automated transmission electron microscopy.

Martin Schorb1, Isabella Haberbosch2,3, Wim J H Hagen4

  • 1Electron Microscopy Core Facility, EMBL, Heidelberg, Germany. martin.schorb@embl.de.

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Summary
This summary is machine-generated.

Automated software tools enhance electron microscopy for structural and cellular biology. This enables high-throughput data collection through image analysis-guided acquisition and feedback microscopy.

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Area of Science:

  • Electron microscopy
  • Cellular biology
  • Structural biology

Background:

  • Increasing demand for high-throughput data collection in electron microscopy.
  • Need for automated acquisition methods in biological imaging.

Purpose of the Study:

  • To present a software combination for automated data acquisition in transmission electron microscopy.
  • To enable feedback microscopy guided by image analysis.

Main Methods:

  • Utilizing SerialEM for microscope and detector control with flexible automation.
  • Integrating Py-EM with SerialEM for specimen-specific image analysis pipelines.
  • Implementing automated tasks at multiple positions for various acquisition schemes.

Main Results:

  • Demonstrated dose reduction in cryo-electron microscopy experiments.
  • Achieved fully automated acquisition of all cells in a plastic section.
  • Enabled automated targeting on serial sections for 3D volume imaging across multiple grids.

Conclusions:

  • The presented software tools facilitate automated, high-throughput data collection in electron microscopy.
  • Image analysis-guided acquisition and feedback microscopy enhance efficiency and scope of biological imaging.
  • The system is versatile for diverse applications including cryo-electron microscopy and 3D volume imaging.