Cryo-X-ray tomography of vaccinia virus membranes and inner compartments.

Vitrified unstained purified vaccinia virus particles have been used as a test sample to evaluate the capabilities of cryo-X-ray tomography. Embedded in a thick layer of vitreous ice, the viral particles representing the mature form of the virus (MV) were visualized using full-field transmission X-ray tomography. The tomographic reconstructions reveal the viral brick-shaped characteristic structures with a size of 250x270x360nm(3). The X-ray tomograms show the presence of a clearly defined external envelope, together with an inner core surrounded by an internal envelope, including areas with clear differential density, which correlate well with those features previously described for these viral particles using electron microscopy analyses. A quantitative assessment of the resolution attained in X-ray and electron tomograms of the viral particles prepared under the same conditions yields values of 25.7 and 6.7nm half-pitch, respectively. Although the resolution of the X-ray microscope is well above the dimensions of the membranous compartments, the strong differential contrast exhibited makes it possible to precisely reveal them without any contrasting reagent within this small and complex biological sample.

[1]  J R Kremer,et al.  Computer visualization of three-dimensional image data using IMOD. , 1996, Journal of structural biology.

[2]  E. Anderson,et al.  Soft X-ray microscopy at a spatial resolution better than 15 nm , 2005, Nature.

[3]  Wolfgang Baumeister,et al.  Cryo-electron tomography of vaccinia virus. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[4]  M. Esteban.  Defective vaccinia virus particles in interferon-treated infected cells. , 1984, Virology.

[5]  Patricia G. Blauner.  High resolution x-ray mask repair , 1995 .

[6]  Jun Liu,et al.  Electron tomography of viruses. , 2007, Current opinion in structural biology.

[7]  R. Wepf,et al.  Structure and Assembly of Intracellular Mature Vaccinia Virus: Isolated-Particle Analysis , 2001, Journal of Virology.

[8]  W. Yun,et al.  Ellipsoidal and parabolic glass capillaries as condensers for x-ray microscopes. , 2008, Applied optics.

[9]  Gareth Griffiths,et al.  Whole Cell Cryo-Electron Tomography Reveals Distinct Disassembly Intermediates of Vaccinia Virus , 2007, PloS one.

[10]  John C. H. Spence,et al.  Experimental High-Resolution Electron Microscopy , 1980 .

[11]  Emil Wolf,et al.  CHAPTER X – INTERFERENCE AND DIFFRACTION WITH PARTIALLY COHERENT LIGHT , 1980 .

[12]  R. Henderson,et al.  Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. , 2003, Journal of molecular biology.

[13]  J. Frank.  Electron tomography : methods for three-dimensional visualization of structures in the cell , 2005 .

[14]  P. Guttmann,et al.  Ellipsoidal capillary as condenser for the BESSY full-field x-ray microscope , 2009 .

[15]  M. L. Le Gros,et al.  X-ray tomography generates 3-D reconstructions of the yeast, saccharomyces cerevisiae, at 60-nm resolution. , 2003, Molecular biology of the cell.

[16]  V. Lučić,et al.  Structural studies by electron tomography: from cells to molecules. , 2005, Annual review of biochemistry.

[17]  Gerry McDermott,et al.  Quantitative 3-D imaging of eukaryotic cells using soft X-ray tomography. , 2008, Journal of structural biology.

[18]  D A Agard,et al.  Toward fully automated high-resolution electron tomography. , 1996, Journal of structural biology.

[19]  Kay Grünewald,et al.  Structure of complex viruses and virus-infected cells by electron cryo tomography. , 2006, Current opinion in microbiology.

[20]  M. L. Le Gros,et al.  X-ray tomography of Schizosaccharomyces pombe. , 2007, Differentiation; research in biological diversity.

[21]  Geoffrey L. Smith,et al.  Vaccinia virus morphogenesis and dissemination. , 2008, Trends in microbiology.

[22]  S. Rehbein,et al.  X‐Ray Microscopy at BESSY: From Nano‐Tomography to Fs‐Imaging , 2007 .

[23]  B. Niemann,et al.  Computed tomography of cryogenic biological specimens based on X-ray microscopic images. , 2000, Ultramicroscopy.

[24]  G. Schneider,et al.  X-ray tomography of a microhabitat of bacteria and other soil colloids with sub-100 nm resolution. , 2003, Micron.

[25]  Paula Traktman,et al.  In a nutshell: structure and assembly of the vaccinia virion. , 2006, Advances in virus research.

[26]  D A Agard,et al.  IVE (Image Visualization Environment): a software platform for all three-dimensional microscopy applications. , 1996, Journal of structural biology.

[27]  M. Esteban,et al.  Humoral immune response elicited by highly attenuated variants of vaccinia virus and by an attenuated recombinant expressing HIV-1 envelope protein. , 1989, Virology.

[28]  G. Schneider,et al.  Cryo X-ray microscopy with high spatial resolution in amplitude and phase contrast. , 1998, Ultramicroscopy.

[29]  M. Esteban,et al.  Generation of a dominant 8-MDa deletion at the left terminus of vaccinia virus DNA. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[30]  J. Dubochet,et al.  Structure of intracellular mature vaccinia virus observed by cryoelectron microscopy , 1994, Journal of virology.

[31]  David A. Agard,et al.  PRIISM: an integrated system for display and analysis of 3-D microscope images , 1992, Electronic Imaging.

[32]  J Bernard Heymann,et al.  Bsoft: image processing and molecular modeling for electron microscopy. , 2007, Journal of structural biology.

[33]  High resolution X-ray tomography with applications in biology and materials science , 2003 .

[34]  G. Griffiths,et al.  Structure and Assembly of Intracellular Mature Vaccinia Virus: Thin-Section Analyses , 2001, Journal of Virology.

[35]  K. Tokuyasu.  A TECHNIQUE FOR ULTRACRYOTOMY OF CELL SUSPENSIONS AND TISSUES , 1973, The Journal of cell biology.

[36]  Erik H. Anderson,et al.  Computed tomography of cryogenic cells , 2001 .

[37]  Giovanni Cardone,et al.  A resolution criterion for electron tomography based on cross-validation. , 2005, Journal of structural biology.

[38]  Gerry McDermott,et al.  X-ray tomography of whole cells. , 2005, Current opinion in structural biology.

[39]  M. Esteban,et al.  Virus attenuation and identification of structural proteins of vaccinia virus that are selectively modified during virus persistence , 1987, Journal of virology.