Histochemistry and Cell Biology

, Volume 138, Issue 4, pp 549–556 | Cite as

FIB/SEM tomography with TEM-like resolution for 3D imaging of high-pressure frozen cells

  • Clarissa Villinger
  • Heiko Gregorius
  • Christine Kranz
  • Katharina Höhn
  • Christin Münzberg
  • Götz von Wichert
  • Boris Mizaikoff
  • Gerhard Wanner
  • Paul Walther
Original Paper

Abstract

Focused ion beam/scanning electron microscopy (FIB/SEM) tomography is a novel powerful approach for three-dimensional (3D) imaging of biological samples. Thereby, a sample is repeatedly milled with the focused ion beam (FIB) and each newly produced block face is imaged with the scanning electron microscope (SEM). This process can be repeated ad libitum in arbitrarily small increments allowing 3D analysis of relatively large volumes such as eukaryotic cells. High-pressure freezing and freeze substitution, on the other hand, are the gold standards for electron microscopic preparation of whole cells. In this work, we combined these methods and substantially improved resolution by using the secondary electron signal for image formation. With this imaging mode, contrast is formed in a very small, well-defined area close to the newly produced surface. By using this approach, small features, so far only visible in transmission electron microscope (TEM) (e.g., the two leaflets of the membrane bi-layer, clathrin coats and cytoskeletal elements), can be resolved directly in the FIB/SEM in the 3D context of whole cells.

Keywords

Focused ion beam Tomography Scanning electron microscopy High-pressure freezing Freeze substitution BON cells FIB/SEM Secondary electrons 

Supplementary material

418_2012_1020_MOESM1_ESM.m4v (26.4 mb)
Video 1: FIB/SEM dataset of Fig. 2 according to Protocol A. A Volume of 25 μm × 19 μm × 6.5 μm is visible in this slice and view dataset. The ultrastructural features such as mitochondria or the Golgi apparatus are well preserved and well resolved (M4V 27,070 kb)
418_2012_1020_MOESM2_ESM.m4v (9.5 mb)
Video 2: FIB/SEM dataset of Fig. 4 according to Protocol B. A volume of about 2 μm × 1.5 μm × 3 μm is visible in this “slice and view” dataset, recorded at high magnification. Note the good visibility of fine structural details such as the two leaflets of the membrane bi-layer and ribosomes (M4V 9,710 kb)

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Clarissa Villinger
    • 1
  • Heiko Gregorius
    • 1
  • Christine Kranz
    • 2
  • Katharina Höhn
    • 1
    • 3
  • Christin Münzberg
    • 4
  • Götz von Wichert
    • 4
  • Boris Mizaikoff
    • 2
  • Gerhard Wanner
    • 5
  • Paul Walther
    • 1
  1. 1.Central Facility for Electron MicroscopyUlm UniversityUlmGermany
  2. 2.Institute of Analytical and Bioanalytical ChemistryUlm UniversityUlmGermany
  3. 3.Department of Infectious Diseases, VirologyUniversity of HeidelbergHeidelbergGermany
  4. 4.Institute for Internal Medicine IUniversity Hospital UlmUlmGermany
  5. 5.Departement Biologie ILudwig-Maximilians-Universität MünchenMunchenGermany

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