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Zooming in and out: Exploring RNA Viral Infections with Multiscale Microscopic Methods.
Viruses
September 2024
Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK.
RNA viruses, being submicroscopic organisms, have intriguing biological makeups and substantially impact human health. Microscopic methods have been utilized for studying RNA viruses at a variety of scales. In order of observation scale from large to small, fluorescence microscopy, cryo-soft X-ray tomography (cryo-SXT), serial cryo-focused ion beam/scanning electron microscopy (cryo-FIB/SEM) volume imaging, cryo-electron tomography (cryo-ET), and cryo-electron microscopy (cryo-EM) single-particle analysis (SPA) have been employed, enabling researchers to explore the intricate world of RNA viruses, their ultrastructure, dynamics, and interactions with host cells.
View Article and Find Full Text PDFCorrelative cryo-microscopy pipelines for in situ cellular studies.
Methods Cell Biol
May 2024
NanoImaging Core Facility, Institut Pasteur, Paris, France. Electronic address:
Correlative cryo-microscopy pipelines combining light and electron microscopy and tomography in cryogenic conditions (cryoCLEM) on the same sample are powerful methods for investigating the structure of specific cellular targets identified by a fluorescent tag within their unperturbed cellular environment. CryoCLEM approaches circumvent one of the inherent limitations of cryo EM, and specifically cryo electron tomography (cryoET), of identifying the imaged structures in the crowded 3D environment of cells. Whereas several cryoCLEM approaches are based on thinning the sample by cryo FIB milling, here we present detailed protocols of two alternative cryoCLEM approaches for in situ studies of adherent cells at the single-cell level without the need for such cryo-thinning.
View Article and Find Full Text PDFThe Potential of Subsampling and Inpainting for Fast Low-Dose Cryo FIB-SEM Imaging.
Microsc Microanal
March 2024
Department of Mechanical, Materials and Aerospace Engineering, University of Liverpool, Liverpool, L69 3BX, UK.
Traditional image acquisition for cryo focused ion-beam scanning electron microscopy (FIB-SEM) tomography often sees thousands of images being captured over a period of many hours, with immense data sets being produced. When imaging beam sensitive materials, these images are often compromised by additional constraints related to beam damage and the devitrification of the material during imaging, which renders data acquisition both costly and unreliable. Subsampling and inpainting are proposed as solutions for both of these aspects, allowing fast and low-dose imaging to take place in the Focused ion-beam scanning electron microscopy FIB-SEM without an appreciable loss in image quality.
View Article and Find Full Text PDFIn Situ Imaging of Virus-Infected Cells by Cryo-Electron Tomography: An Overview.
Subcell Biochem
January 2024
MRC-University of Glasgow Centre for Virus Research, Sir Michael Stoker Building, Garscube Campus, Glasgow, Scotland, UK.
Cryo-electron tomography (cryo-ET) has emerged as a powerful tool in structural biology to study viruses and is undergoing a resolution revolution. Enveloped viruses comprise several RNA and DNA pleomorphic viruses that are pathogens of clinical importance to humans and animals. Considerable efforts in cryogenic correlative light and electron microscopy (cryo-CLEM), cryogenic focused ion beam milling (cryo-FIB), and integrative structural techniques are helping to identify virus structures within cells leading to a rise of in situ discoveries shedding light on how viruses interact with their hosts during different stages of infection.
View Article and Find Full Text PDFCorrelative Light and Electron Cryo-Microscopy Workflow Combining Micropatterning, Ice Shield, and an In-Chamber Fluorescence Light Microscope.
Bio Protoc
December 2023
Ernst-Ruska Centre for Microscopy and Spectroscopy with Electrons, ER-C-3/Structural Biology, Forschungszentrum Jülich, Jülich, Germany.
In situ cryo-electron tomography (cryo-ET) is the most current, state-of-the-art technique to study cell machinery in its hydrated near-native state. The method provides ultrastructural details at sub-nanometer resolution for many components within the cellular context. Making use of recent advances in sample preparation techniques and combining this method with correlative light and electron microscopy (CLEM) approaches have enabled targeted molecular visualization.
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