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Reconstruction

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Display image description: Single computed slice through a dual axis tilt electron tomographic reconstruction of a freeze-substituted VeroE6 cultured cell infected with the SARS-CoV.

Full resolution image description: Tomographic volume in IMOD format (6022_Suppmovie3.rec) of the infected VeroE6 cells.

Volume_dimension: 2023, 2023, 167

Volume scale: 0.0012, 0.0012, 0.0012

Animation description: Animation (6022.avi) through the tomographic volume of freeze-substituted VeroE6 culture cells infected with SARS-CoV.

Image 2D

Display image description: Single tilt image from a double tilt series through 0.2 um thick section of a freeze-substituted VeroE6 cultured cell infected with the SARS-CoV taken with transmission electron microscopy. Dark particles are 10 nm colloidal gold particles that were applied to the surface to serve as fiducial markers for alignment of the tilt series.

Full resolution image description: Aligned tilt images (6022_Suppmovie3.ali) in IMOD format

License

Attribution Only: This image is licensed under a Creative Commons Attribution License. View License Deed | View Legal Code

CCDB:6022^* Cite

Project: P2005

Project name: Membrane Modifications Induced by Corona Virus

Description: Electron tomography of African Green Monkey epithelial cells infected with SARS-coronavirus

Funding agency: Leiden University Medical Center (LUMC, the Netherlands), Council for Chemical Sciences of the Netherlands Organization for Scientific Research (NOW-CW grants 700.52.306 and 700.55.002), European Commission under context of Euro-Asian SARS-DTV Network (SP22-CT-2004-511064)

Leader(s): Kèvin Knoops; Abraham J. Koster; A. Mieke Mommaas and Eric J. Snijder

Collaborator(s): Marjolein Kikkert; Sjoerd H.E. van den Worm; Jessika C. Zevenhoven-Dobbe; Yvonne van der Meer

Start date: 02-01-2005

End date: 02-01-2005

Experiment

Experiment ID: 6014

Experiment date: 06-22-2006

Title: Electron Tomography of Membrane Modification

Purpose: Study the membrane modification of African Green Monkey epithelial cells post-infection of SARS-coronavirus

Experimenter(s): Kevin Knoops

Microscopy product

Microscopy product ID: 6022

Instrument: FEI Tecnai 12

Microscopy type: TEM

Product type: DOUBLE TILT

Image basename: suppmovie3

Spatial Axis	Image Size	Pixel Size
X	2023px	1.2 nm/pixels
Y	2023px	1.2 nm/pixels

Subject

Species: monkey

Scientific name: Cercopithecus aethiops

Strain: Vervet

Group by: time after infection

Treatment: 7 hrs post-infection with SARS-CoV strain Frankfurt-1 (provided by Dr. H. F. Rabenau 603 and Dr. H. W. Doerr (Johann-Wolfgang-Goethe-Universität, Frankfurt am Main, Germany)

Age class: n/a

Tissue section

Anatomical location: epithelium

Microtome: Leica UC6 ultramicrotome

Thickness: 0.2 µm

Specimen description

Organ: kidney

Cell type: VeroE6

Imaging parameters

Type: Electron microscopy product

Recording medium: Slow scan cooled 2K CCD camera

Magification: 18500

Accelerating voltage: 120 kV

Specimen preparation

Protocol used: For ultrastructural morphological investigations, SARS-CoV-infected Vero E6 cells were pre-fixed (for biosafety reasons) overnight with 3% paraformaldehyde in 0.1M PHEM buffer (60 mM piperazide-1,4-bis[2-ethanesulfonic acid], 25 mM HEPES, 2 mM MgCl2, 10 mM EGTA) at various time points after infection. For cryo-fixation, cell monolayers adhered to Thermanox coverslips (Nunc, Denmark) were plunged into liquid ethane. Freeze substitution was performed at -90C in an automated freeze-substitution system (Leica, Austria) using an FS medium consisting of 90% acetone and 10% water, containing 1% osmium tetroxide and 0.5 % uranyl acetate. After washing with pure acetone at RT, the samples were embedded in epoxy LX-12 resin. Thin sections were collected on 100 Mesh grids, 1.25% pioloform support film contrasted with uranyl acetate and lead hydroxide and subsequently viewed at 80 kV with a Philips CM-10 transmission electron microscope (Philips, The Netherlands).

Imaging product type

Type: Double tilt

X min range: -65 degrees

X max range: 65 degrees

X tilt increment: 1 degrees

Y min range: -65 degrees

Y max range: 65 degrees

Y tilt increment: 1 degrees

Description: specimens were rotated 90 deg around the Z-axis using a dual-axis tilt tomography holder (Fishione L