Name: Serie: Focused Ion Beam Scanning Electron Microscopy for 3D Reconstruction of Synaptically Linked Neurons - Experiment
Uploaded: 2025-05-30T09:07:31.000+00:00
Duration: 1 min 21 s
Description: 31 Aufrufe. Focused Ion Beam Scanning Electron Microscopy for 3D Reconstruction of Synaptically Linked Neurons

focused ion beam scanning electron microscopy for 3d reconstruction of synaptically linked neurons

Focused Ion Beam Scanning Electron Microscopy for 3D Reconstruction of Synaptically Linked Neurons

Source:&#160;Maco B,&#160;et al. Correlative&#160;In Vivo 2 Photon and Focused Ion Beam Scanning Electron Microscopy of Cortical Neurons.&#160;PLoS ...

Take a fixed section of mouse cortical brain tissue exhibiting neuronal axons and dendrites. The dendrites possess protrusions, known as dendritic spines, which form synapses with axons. Incubate the tissue with osmium-ferrocyanide, which binds to lipids, followed by another osmium application, which enhances structural contrast for imaging. Stain with a heavy metal compound that binds to macromolecules and enhances contrast. Dehydrate using increasing alcohol concentrations for resin embedding. Embed the tissue in resin, mount it on a holder, and coat it with gold to optimize imaging quality. Use focused ion beam scanning electron microscopy for three-dimensional imaging. Employ an ion beam to remove a thin layer of resin-embedded tissue, exposing an imaging surface. Focus an electron beam on the exposed surface and detect the backscattered electrons to generate an image. &#160;&#160; &#160; Perform serial slicing and imaging and reconstruct a three-dimensional image, revealing a synaptically linked cortical neuronal network.&#160;&#160;

focused-ion-beam-scanning-electron-microscopy-for-3d-reconstruction

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuroimaging

Synaptic & Cellular Imaging

31 Aufrufe. Focused Ion Beam Scanning Electron Microscopy for 3D Reconstruction of Synaptically Linked Neurons

Serie: Focused Ion Beam Scanning Electron Microscopy for 3D Reconstruction of Synaptically Linked Neurons - Experiment

A Method for Obtaining Serial Ultrathin Sections of Microorganisms in Transmission Electron Microscopy

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin sections of the specimen. Although preparing serial ultrathin sections is considered to be very difficult, it is rather easy if the proper method is used. In this paper, we show a step-by-step procedure for safely obtaining serial ultrathin sections of microorganisms. The key points of this method are: 1) to use the large part of the specimen and adjust the specimen surface and knife edge so that they are parallel to each other; 2) to cut serial sections in groups and avoid difficulty in separating sections using a pair of hair strands when retrieving a group of serial sections onto the slit grids; 3) to use a 'Section-holding loop' and avoid mixing up the order of the section groups; 4) to use a 'Water-surface-raising loop' and make sure the sections are positioned on the apex of the water and that they touch the grid first, in order to place them in the desired position on the grids; 5) to use the support film on an aluminum rack and make it easier to recover the sections on the grids and to avoid wrinkling of the support film; and 6) to use a staining tube and avoid accidentally breaking the support films with tweezers. This new method enables obtaining serial ultrathin sections without difficulty. The method makes it possible to analyze cell structures of microorganisms at high resolution in 3D, which cannot be achieved by using the automatic tape-collecting ultramicrotome method and serial block-face or focused ion beam scanning electron microscopy.

This study presents reliable and easy procedures for obtaining serial ultrathin sections of a microorganism without expensive equipment in transmission electron microscopy.

Eine Methode für den Erhalt der ultradünnen Serienschnitte von Mikroorganismen im Transmissions-Elektronenmikroskopie

Observing cells and cell components in three dimensions at high magnification in transmission electron microscopy requires preparing serial ultrathin ...

Forschung

JoVE Journal

Ingenieurwesen

Mitochondria and Endoplasmic Reticulum Imaging by Correlative Light and Volume Electron Microscopy

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved structures are folded into various shapes to form a dynamic network depending on the cellular conditions. Visualization of this network between mitochondria and ER has been attempted using super-resolution fluorescence imaging and light microscopy; however, the limited resolution is insufficient to observe the membranes between the mitochondria and ER in detail. Transmission electron microscopy provides good membrane contrast and nanometer-scale resolution for the observation of cellular organelles; however, it is exceptionally time-consuming when assessing the three-dimensional (3D) structure of highly curved organelles. Therefore, we observed the morphology of mitochondria and ER via correlative light-electron microscopy (CLEM) and volume electron microscopy techniques using enhanced ascorbate peroxidase 2 and horseradish peroxidase staining. An en bloc staining method, ultrathin serial sectioning (array tomography), and volume electron microscopy were applied to observe the 3D structure. In this protocol, we suggest a combination of CLEM and 3D electron microscopy to perform detailed structural studies of mitochondria and ER.

We present a protocol to study the distribution of mitochondria and endoplasmic reticulum in whole cells after genetic modification using correlative light and volume electron microscopy including ascorbate peroxidase 2 and horseradish peroxidase staining, serial sectioning of cells with and without the target gene in the same section, and serial imaging via electron microscopy.

Mitochondrien und Endoplasmatische Retikulum-Bildgebung durch korrelative Licht- und Volumenelektronenmikroskopie

Cellular organelles, such as mitochondria and endoplasmic reticulum (ER), create a network to perform a variety of functions. These highly curved ...

Biochemie

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

Miniaturisierte Probenvorbereitung für Transmissions-Elektronenmikroskopie

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

Focused Ion Beam Scanning Electron Microscopy for 3D Reconstruction of Synaptically Linked Neurons

Transkript

Eine Methode für den Erhalt der ultradünnen Serienschnitte von Mikroorganismen im Transmissions-Elektronenmikroskopie

Mitochondrien und Endoplasmatische Retikulum-Bildgebung durch korrelative Licht- und Volumenelektronenmikroskopie

Miniaturisierte Probenvorbereitung für Transmissions-Elektronenmikroskopie