title: Cryo X-ray nano-tomography of vaccinia virus infected cells.
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.
authors.affiliation: Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas, Campus Cantoblanco, 28049 Madrid, Spain.
authors.forename: Francisco Javier
authors.initials: FJ
authors.lastname: Chichón
authors.forename: Maria Josefa
authors.initials: MJ
authors.lastname: Rodríguez
authors.forename: Eva
authors.initials: E
authors.lastname: Pereiro
authors.forename: Michele
authors.initials: M
authors.lastname: Chiappi
authors.forename: Beatriz
authors.initials: B
authors.lastname: Perdiguero
authors.forename: Peter
authors.initials: P
authors.lastname: Guttmann
authors.forename: Stephan
authors.initials: S
authors.lastname: Werner
authors.forename: Stefan
authors.initials: S
authors.lastname: Rehbein
authors.forename: Gerd
authors.initials: G
authors.lastname: Schneider
authors.forename: Mariano
authors.initials: M
authors.lastname: Esteban
authors.forename: José L
authors.initials: JL
authors.lastname: Carrascosa
mesh_terms: D000818:Animals; D002460:Cell Line; D048430:Cell Shape; D002642:Chick Embryo; D015925:Cryopreservation / Q000379:methods; D022083:Cytoplasmic Structures / Q000648:ultrastructure / Q000821:virology; D054884:Host-Pathogen Interactions; D008853:Microscopy / Q000379:methods*; D059010:Single-Cell Analysis; D014056:Tomography, X-Ray / Q000379:methods*; D014616:Vaccinia virus / Q000502:physiology* / Q000648:ultrastructure; D014771:Virion / Q000648:ultrastructure