Cryo X-ray nano-tomography of vaccinia virus infected cells

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 b...

Full description

Saved in:
Bibliographic Details
Published in:Journal of structural biology 2012-02, Vol.177 (2), p.202-211
Main Authors: Chichón, Francisco Javier, Rodríguez, Maria Josefa, Pereiro, Eva, Chiappi, Michele, Perdiguero, Beatriz, Guttmann, Peter, Werner, Stephan, Rehbein, Stefan, Schneider, Gerd, Esteban, Mariano, Carrascosa, José L.
Format: Article
Language:English
Subjects:
Animals
Cell Line
Cell Shape
Chick Embryo
Correlative microscopy
Cryo X-ray nano-tomography
Cryopreservation - methods
Cytoplasmic Structures - ultrastructure
Cytoplasmic Structures - virology
Electron tomography
Host-Pathogen Interactions
Microscopy - methods
Radiation damage
Single-Cell Analysis
Tomography, X-Ray - methods
Vaccinia virus
Vaccinia virus - physiology
Vaccinia virus - ultrastructure
Virion - ultrastructure
Virus assembly
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:1047-8477
1095-8657
DOI:10.1016/j.jsb.2011.12.001