Core Facility for Electron Tomography

 To understand the life processes it is necessary to know where the specific macromolecules are located within a cell and tissue and how the macromolecules interact with each other. Electron tomography permits subcellular and macromolecular assemblies to be studied in three dimensions; the molecular objects are reconstructed in three dimensions from a series of electron micrographs representing different projections of the objects.

Images: Sampling of projection images (A) and 3D-reconstruction by backprojection (B).

Reproduced from:

Electron tomography of molecules and cells.
Baumeister W, Grimm R, Walz J
Trends Cell Biol. 1999 Feb;9(2):81-5

The possibility to directly image individual biochemically isolated molecular objects provides information on how the various molecules/assemblies change their conformation (dynamics) and how they bind to each other. As the molecular complexes can also be visualized as they appear within the cell, it is possible to determine where they are located and how the macromolecules interact with each other.

 

 

Movie: 3D architecture of the presynaptic cytosolic projection in a Drosophila neuromuscular junction. The tomogram is first presented separately and then related to a 3D model of the projection. Central core in magenta, extensions in green and connected synaptic vesicles in blue.

From:

Two pools of vesicles associated with the presynaptic cytosolic projection in Drosophila neuromuscular junctions.
Jiao W, Masich S, Franzén O, Shupliakov O
J. Struct. Biol. 2010 Dec;172(3):389-94

The method is technically advanced and requires both expert competence and expensive, sophisticated equipment, and the studies have to be carried out with the support of an electron tomography facility.

Welcome to the Core Facility for Electron Tomography at KI! Can we help you with your research?

Instruments available

  • Transmission electron microscope CM200 equipped with a cooled slow scan 2048x2048 TemCam- F224 CCD camera (pixel size 24 µm) and software for automated data collection (TVIPS, Gauting, Germany)
  • Transmission electron microscope CM120 equipped with a side-entry CCD Olympus MageView III camera
  • High pressure freezer EMPACT2 (Leica)
  • Equipment for freeze substitution AFS
  • Carbon coater
  • Glow-discharge unit
  • Ultramicrotome
  • Cryo-ultramicrotome

Service offered

  • Preparation of cryo-specimens
  • Preparation of negatively stained specimens
  • Teaching to operate TEM for independent data collection
  • Acquisition of 2D-images
  • Acquisition of tilt-series for electron tomography
  • Reconstruction of tomography data

Cryoelectron tomography of a DNA origami structure

Left image: Cryoelectron microscopic image of bunny-shaped DNA origamis. The origami structure corresponds to a 3D mesh shaped into a bunny and rendered in DNA (insert). The scale bar corresponds to 100 nm.

Right image: Analysis of a tomogram of  a single bunny-shaped DNA origami. Top figure: 3D rendering of the origami structure rotated to correspond to the tomogram. Below, three progressive slices in the 3D reconstruction are displayed; insets show the expected outlines from the corresponding sections of the digital model.

Movie displaying consecutive slices through a 3D reconstruction (tomogram) of a specimen with bunny-shaped DNA origamis.

From:

DNA rendering of polyhedral meshes at the nanoscale.
Benson E, Mohammed A, Gardell J, Masich S, Czeizler E, Orponen P, et al
Nature 2015 Jul;523(7561):441-4

Teaching and training

We give a course in “Basic transmission electron microscopy in cell biology” (approximately once every second year). Organizers: Prof. Oleg Shupliakov and Dr. Sergej Masich.

For those who want to use our microscopes or other equipment for independent data collection, we provide individual introductory or advanced training

Prices for service (SEK/hour)

  Karolinska Institutet Other universities Industry
CM120 microscopy 500 600 800
CM200 microscopy 600 700 1000
Expert support 500 600 750

Selected publications

Recently published studies conducted with the support of the Electron Tomography Facility at KI:

DNA rendering of polyhedral meshes at the nanoscale.
Benson E, Mohammed A, Gardell J, Masich S, Czeizler E, Orponen P, et al
Nature 2015 Jul;523(7561):441-4

Skin Lamellar Bodies are not Discrete Vesicles but Part of a Tubuloreticular Network.
den Hollander L, Han H, de Winter M, Svensson L, Masich S, Daneholt B, et al
Acta Derm. Venereol. 2016 Mar;96(3):303-8

The central element of the synaptonemal complex in mice is organized as a bilayered junction structure.
Hernández-Hernández A, Masich S, Fukuda T, Kouznetsova A, Sandin S, Daneholt B, et al
J. Cell. Sci. 2016 Jun;129(11):2239-49

More reading

Tutorials in Transmission Electron Microscopy
–  website by Prof. John Rodenburg, University of Sheffield.

Electron tomography of molecules and cells.
Baumeister W, Grimm R, Walz J
Trends Cell Biol. 1999 Feb;9(2):81-5

Electron Cryotomography
Murphy GE and Jensen GJ
BioTechniques (2007) Vol. 43 No. 4

Cryo-Electron Tomography: Can it Reveal the Molecular Sociology of Cells in Atomic Detail?
Beck M, Baumeister W
Trends Cell Biol. 2016 Nov;26(11):825-837

Organisation and support

The Electron Tomography Facility is supported by Karolinska Institutet and the Stockholm County Council, through the Joint Core Facility Committee.

Head of facility: Prof. em. Bertil Daneholt, Department of Cell and Molecular Biology, KI

Manager: Dr. Sergej Masich, Department of Cell and Molecular Biology, KI

Advisory committee:
Prof. em. Bertil Daneholt (convener)
Assoc. prof. Björn Högberg, Department of Medical Biochemistry and Biophysics, KI
Prof. Oleg Shupliakov, Department of Neuroscience, KI
Prof. Mats Wahlgren, Department of Microbiology, Tumor and Cell Biology, KI
Assoc. prof. Ozan Öktem, Department of Mathematics, KTH.

Contact us

PhD, Senior lab manager

Sergej Masich

Phone: +46 (0)8-524 87361
E-mail: Sergej.Masich@ki.se
Address: CMB, von Eulers väg 1
SE17177 Stockholm, Sweden

The facility is currently located at the Department of Cell and Molecular Biology, von Eulers väg 1, KI, Campus Solna.

Core facilityImagingMicroscopy