Cryo X-ray Nano-tomography of Vaccinia Virus Infected Cells

Overview

Journal J Struct Biol

Specialties Cell Biology
Molecular Biology

Date 2011 Dec 20

PMID 22178221

Citations 30

Authors

Francisco Javier Chichon

Maria Josefa Rodriguez

Eva Pereiro

Michele Chiappi

Beatriz Perdiguero

Peter Guttmann

Stephan Werner

Stefan Rehbein

Gerd Schneider

Mariano Esteban

Jose L Carrascosa

Affiliations

Soon will be listed here.

Abstract

We have performed full-field cryo X-ray microscopy in the water window photon energy range on vaccinia virus (VACV) infected cells to produce tomographic reconstructions. PtK2 cells were infected with a GFP-expressing VACV strain and frozen by plunge fast freezing. The infected cells were selected by light fluorescence microscopy of the GFP marker and subsequently imaged in the X-ray microscope under cryogenic conditions. Tomographic tilt series of X-ray images were used to yield three-dimensional reconstructions showing different cell organelles (nuclei, mitochondria, filaments), together with other structures derived from the virus infection. Among them, it was possible to detect viral factories and two types of viral particles related to different maturation steps of VACV (immature and mature particles), which were compared to images obtained by standard electron microscopy of the same type of cells. In addition, the effect of radiation damage during X-ray tomographic acquisition was analyzed. Thin sections studied by electron microscopy revealed that the morphological features of the cells do not present noticeable changes after irradiation. Our findings show that cryo X-ray nano-tomography is a powerful tool for collecting three-dimensional structural information from frozen, unfixed, unstained whole cells with sufficient resolution to detect different virus particles exhibiting distinct maturation levels.

Citing Articles

3D Cryo-Correlative Methods to Study Virus Structure and Dynamics Within Cells.

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Soft X-ray Microscopy in Cell Biology: Current Status, Contributions and Prospects.

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Correlative Cryo-imaging Using Soft X-Ray Tomography for the Study of Virus Biology in Cells and Tissues.

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In Situ Imaging of Virus-Infected Cells by Cryo-Electron Tomography: An Overview.

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3D surface reconstruction of cellular cryo-soft X-ray microscopy tomograms using semisupervised deep learning.

Dyhr M, Sadeghi M, Moynova R, Knappe C, Kepsutlu Cakmak B, Werner S Proc Natl Acad Sci U S A. 2023; 120(24):e2209938120.

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