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Cryo-electron Microscopy01:28

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

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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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Immunogold Electron Microscopy01:20

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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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Investigating the Cytoskeleton of DRGs Using Cryo-Electron Microscopy and Deep Learning.

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Summary
This summary is machine-generated.

This study presents a cryogenic electron microscopy (cryo-EM) method for visualizing the ultrastructure of dorsal root ganglion (DRG) neurons. The technique offers a near-native view of axonal organization and cytoskeletal elements, aiding in age-related morphology studies.

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Cryo‐electron tomographyactinaxonal cytoskeletondeep‐learningdorsal root ganglionmicrotubulesneural networkssegmentation

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Area of Science:

  • Neuroscience
  • Cell Biology
  • Microscopy

Background:

  • Classic ultramicroscopy methods for studying neuronal ultrastructure rely on fixation and heavy metal staining, which can introduce artifacts.
  • Cryogenic electron microscopy (cryo-EM) offers a near-native imaging approach, preserving biological specimens without chemical fixation or staining.
  • Axons and varicosities of dorsal root ganglion (DRG) neurons are crucial for sensory information transmission and are amenable to cryo-EM due to their thin structure.

Purpose of the Study:

  • To establish a detailed protocol for examining the ultrastructural organization of cultured DRG neurons using cryo-electron tomography (cryo-ET).
  • To demonstrate the utility of cryo-EM for analyzing age-related changes in axonal morphology.
  • To integrate deep-learning strategies for efficient semi-automated tomographic segmentation and quantitative analysis.

Main Methods:

  • Isolation and culturing of DRG neurons from animals of various ages.
  • Cryo-preservation of cultured neurons for cryo-EM sample preparation.
  • High-resolution cryo-electron tomography data acquisition.
  • Deep-learning-assisted semi-automated segmentation of cytoskeletal elements within axons and varicosities.

Main Results:

  • Cryo-EM successfully visualized the ultrastructural organization of axons and varicosities in near-native conditions.
  • The protocol allows for detailed analysis of cytoskeletal elements, including dimensions and proximity.
  • Segmentations highlighted differences in axonal morphology between young and old DRG neurons.

Conclusions:

  • Cryo-EM provides a powerful, artifact-minimized method for ultrastructural analysis of neuronal processes like axons and varicosities.
  • This technique is particularly valuable for studying age-related changes in neuronal morphology.
  • The integration of deep learning enhances the efficiency and quantitative capabilities of cryo-EM analysis in neuroscience research.