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Histochemistry and Cell Biology

, Volume 135, Issue 1, pp 1–9 | Cite as

Preparation of cryofixed cells for improved 3D ultrastructure with scanning transmission electron tomography

  • Katharina Höhn
  • Michaela Sailer
  • Li Wang
  • Myriam Lorenz
  • Marion E. Schneider
  • Paul WaltherEmail author
Original Paper

Abstract

Scanning transmission electron tomography offers enhanced contrast compared to regular transmission electron microscopy, and thicker samples, up to 1 μm or more, can be analyzed, since the depth of focus and inelastic scattering are not limitations. In this study, we combine this novel imaging approach with state of the art specimen preparation by using novel light transparent sapphire specimen carrier for high-pressure freezing and a freeze substitution protocol for better contrast of membranes. This combination allows for imaging membranes and other subcellular structures with unsurpassed quality. This is demonstrated with mitochondria, where the inner and outer mitochondrial membranes as well as the membranes in the cristae appear in very close apposition with a minimal intermembrane space. These findings correspond well with old observations using freeze fracturing. In 880-nm thick sections of hemophagocytes, the three-dimensional structure of membrane sheets could be observed in the virtual sections of the tomogram. Microtubules, actin and intermediate filaments could be visualized within one sample. Intermediate filaments, however, could even be better observed in 3D using surface scanning electron tomography.

Keywords

STEM tomography High-pressure freezing Membrane Cytoskeleton Mitochondrium Autophagosome Autophagy 

Notes

Acknowledgments

We thank Johannes Biskupek for help with the Titan electron microscope and especially for the parallel beam alignment, Eberhard Schmid for excellent technical support and for developing the important procedure of properly mounting the thick sections on the special copper grids, and Ganesh V. Pusapati for help with the cell cultures. This work was supported by the DFG Sonderforschungsbereich 518, project A15 and B21, by the DFG Einzelantrag WA 1458/3-1 and by The Histiocytosis Association of America.

Supplementary material

Supplementary material 1 (MOV 4,413 kb)

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Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Katharina Höhn
    • 1
  • Michaela Sailer
    • 1
  • Li Wang
    • 1
    • 2
  • Myriam Lorenz
    • 3
  • Marion E. Schneider
    • 3
  • Paul Walther
    • 1
    Email author
  1. 1.Electron Microscopy FacilityUlm UniversityUlmGermany
  2. 2.Institute of Virology University Hospital UlmUlmGermany
  3. 3.Section of Experimental AnesthesiologyUniversity Hospital UlmUlmGermany

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