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High-Pressure Freezing for Scanning Transmission Electron Tomography Analysis of Cellular Organelles

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Cell Imaging Techniques

Part of the book series: Methods in Molecular Biology ((MIMB,volume 931))

Abstract

Using an electron microscope’s scanning transmission mode (STEM) for collection of tomographic datasets is advantageous compared to bright field transmission electron microscopic (TEM). For image formation, inelastic scattering does not cause chromatic aberration, since in STEM mode no image forming lenses are used after the beam has passed the sample, in contrast to regular TEM. Therefore, thicker samples can be imaged. It has been experimentally demonstrated that STEM is superior to TEM and energy filtered TEM for tomography of samples as thick as 1 μm. Even when using the best electron microscope, adequate sample preparation is the key for interpretable results. We adapted protocols for high-pressure freezing of cultivated cells from a physiological state. In this chapter, we describe optimized high-pressure freezing and freeze substitution protocols for STEM tomography in order to obtain high membrane contrast.

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References

  1. Baumeister W (2004) Mapping molecular landscapes inside cells. Biol Chem 385:865–872

    Article  PubMed  CAS  Google Scholar 

  2. Hoppe W et al (1974) Three-dimensional reconstruction of individual negatively stained yeast fatty-acid synthetase molecules from tilt series in the electron microscope. Z Physiol Chem 355:1483–1487

    CAS  Google Scholar 

  3. Midgley PA, Weyland M, Thomas JM, Johnson BFG (2001) Z-Contrast tomography: a technique in three-dimensional nanostructural analysis based on Rutherford scattering. Chem Commun 10:907–908

    Article  Google Scholar 

  4. Yakushevska AE et al (2007) STEM tomography in cell biology. J Struct Biol 159:381–391

    Article  PubMed  CAS  Google Scholar 

  5. Aoyama K et al (2008) STEM tomography for thick biological specimens. Ultramicroscopy 109:70–80

    Article  PubMed  CAS  Google Scholar 

  6. Biskupek J et al (2010) Optimization of STEM tomography acquisition—a comparison of convergent beam and parallel beam STEM tomography. Ultramicroscopy 110:1231–1237

    Article  PubMed  CAS  Google Scholar 

  7. Hohmann-Marriott MF et al (2009) Nanoscale 3D cellular imaging by axial scanning transmission electron tomography. Nat Methods 6:729–731

    Article  PubMed  CAS  Google Scholar 

  8. Hawes P et al (2007) Rapid freeze-substitution preserves membranes in high-pressure frozen tissue culture cells. J Microsc 226:182–189

    Article  PubMed  CAS  Google Scholar 

  9. Höhn K et al (2011) Preparation of cryofixed cells for improved 3D ultrastructure with scanning transmission electron tomography. Histochem Cell Biol 135:1–9

    Article  PubMed  Google Scholar 

  10. Walther P, Ziegler A (2002) Freeze substitution of high-pressure frozen samples: the visibility of biological membranes is improved when the substitution medium contains water. J Microsc 208:3–10

    Article  PubMed  CAS  Google Scholar 

  11. Buser C, Walther P (2008) Freeze-substitution: the addition of water to polar solvents enhances the retention of structure and acts at temperatures around −60 °C. J Microsc 230:268–277

    Article  PubMed  CAS  Google Scholar 

  12. Mastronarde DN (2005) Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152:36–51

    Article  PubMed  Google Scholar 

  13. Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116:71–76

    Article  PubMed  CAS  Google Scholar 

  14. Studer D, Michel M, Müller M (1989) High pressure freezing comes of age. Scanning Microsc Suppl 3:253–268

    PubMed  CAS  Google Scholar 

  15. Hohenberg H, Mannweiler K, Müller M (1994) High-pressure freezing of cell suspensions in cellulose capillary tubes. J Microsc 175:34–43

    Article  PubMed  CAS  Google Scholar 

  16. Shimoni E, Müller M (1998) On optimizing high-pressure freezing: from heat transfer theory to a new microbiopsy device. J Microsc 192:236–247

    Article  PubMed  CAS  Google Scholar 

  17. Welsch S et al (2009) Composition and three-dimensional architecture of the dengue virus replication and assembly sites. Cell Host Microbe 5:365–375

    Article  PubMed  CAS  Google Scholar 

  18. Walther P, Wang L, Höhn K (2011) The structure of mitochondria, a study using high-pressure freezing and STEM tomography. Microsc Microanal 17(suppl 2):206–207

    Article  Google Scholar 

  19. McDonald KL, Webb RI (2011) Freeze substitution in 3 hours or less. J Microsc 243:227–233

    Article  PubMed  CAS  Google Scholar 

  20. Walther P et al (1995) Double layer coating for high resolution low temperature SEM. J Microsc 179:229–237

    Article  PubMed  CAS  Google Scholar 

  21. Knott G, Rosset S, Cantoni M (2011) Focussed ion beam milling and scanning electron microscopy of brain tissue. J Vis Exp 53:2588. doi: 10.3791/2588

    PubMed  Google Scholar 

  22. Denk W, Horstmann H (2004) Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol 2:e329

    Article  PubMed  Google Scholar 

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Acknowledgment

We thank Li Wang, Ulm University for providing the sample shown in the figure.

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Authors and Affiliations

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

    Paul Walther, Eberhard Schmid & Katharina Höhn

Authors
  1. Paul Walther
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  2. Eberhard Schmid
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  3. Katharina Höhn
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Corresponding author

Correspondence to Paul Walther .

Editor information

Editors and Affiliations

  1. College of Medicine, Professor, Department of Pathology, University of Vermont, Beaumont Ave 89, Burlington, 05405-1742, Vermont, USA

    Douglas J. Taatjes

  2. , Abt. Zell- und Molekularpathologie, Universität Zürich, Schmelzbergstr. 12, Zürich, 8091, Switzerland

    Jürgen Roth

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Walther, P., Schmid, E., Höhn, K. (2012). High-Pressure Freezing for Scanning Transmission Electron Tomography Analysis of Cellular Organelles. In: Taatjes, D., Roth, J. (eds) Cell Imaging Techniques. Methods in Molecular Biology, vol 931. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-056-4_28

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  • DOI: https://doi.org/10.1007/978-1-62703-056-4_28

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-055-7

  • Online ISBN: 978-1-62703-056-4

  • eBook Packages: Springer Protocols

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