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Electron Microscopy pp 617–638Cite as

Three-Dimensional Imaging of Adherent Cells using FIB/SEM and STEM

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1117))

Abstract

In this chapter we describe three different approaches for three-dimensional imaging of electron microscopic samples: serial sectioning transmission electron microscopy (TEM), scanning transmission electron microscopy (STEM) tomography, and focused ion beam/scanning electron microscopy (FIB/SEM) tomography. With these methods, relatively large volumes of resin-embedded biological structures can be analyzed at resolutions of a few nm within a reasonable expenditure of time. The traditional method is serial sectioning and imaging the same area in all sections. Another method is TEM tomography that involves tilting a section in the electron beam and then reconstruction of the volume by back projection of the images. When the scanning transmission (STEM) mode is used, thicker sections (up to 1 μm) can be analyzed. The third approach presented here is focused ion beam/scanning electron microscopy (FIB/SEM) tomography, in which a sample is repeatedly milled with a 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 arbitrary small increments allowing 3D analysis of relatively large volumes such as eukaryotic cells. We show that resolution of this approach is considerably improved when the secondary electron signal is used. However, the most important prerequisite for three-dimensional imaging is good specimen preparation. For all three imaging methods, cryo-fixed (high-pressure frozen) and freeze-substituted samples have been used.

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Acknowledgments

We thank Renate Kunz, Reinhard Weih, and Eberhard Schmid for their excellent technical assistance. We thank Johannes Biskupek for helping with the Titan electron microscope and especially for the parallel beam alignment. This project was partially supported by the BMBF project NanoCombine. CK acknowledges the support of FEI Co. (Eindhoven, the Netherlands), the German Science Foundation (INST40/385-F1UG), and the Struktur- und Innovationsfonds Baden-Wuerttemberg.

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Authors and Affiliations

  1. Central Facility for Electron Microscopy, Ulm University, Ulm, Germany

    Clarissa Villinger, Martin Schauflinger, Heiko Gregorius, Soufi Nafeey & Paul Walther

  2. Institute of Virology, University Medical Center Ulm, Ulm, Germany

    Clarissa Villinger & Martin Schauflinger

  3. Institute of Analytical and Bioanalytical Chemistry, Ulm University, Ulm, Germany

    Christine Kranz

  4. Department of Infectious Diseases, Virology, University of Heidelberg, Heidelberg, Germany

    Katharina Höhn

Authors
  1. Clarissa Villinger
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  2. Martin Schauflinger
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  3. Heiko Gregorius
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  4. Christine Kranz
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  5. Katharina Höhn
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  6. Soufi Nafeey
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  7. Paul Walther
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Editor information

Editors and Affiliations

  1. University of Western Australia, Crawley, Australia

    John Kuo

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Villinger, C. et al. (2014). Three-Dimensional Imaging of Adherent Cells using FIB/SEM and STEM. In: Kuo, J. (eds) Electron Microscopy. Methods in Molecular Biology, vol 1117. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-776-1_27

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  • DOI: https://doi.org/10.1007/978-1-62703-776-1_27

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-775-4

  • Online ISBN: 978-1-62703-776-1

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