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Lamellar body ultrastructure revisited: high-pressure freezing and cryo-electron microscopy of vitreous sections

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Abstract

Lamellar bodies are the storage sites for lung surfactant within type II alveolar epithelial cells. The structure–function models of lamellar bodies are based on microscopic analyses of chemically fixed tissue. Despite available alternative fixation methods that are less prone to artifacts, such as cryofixation by high-pressure freezing, the nature of the lung, being mostly air filled, makes it difficult to take advantage of these improved methods. In this paper, we propose a new approach and show for the first time the ultrastructure of intracellular lamellar bodies based on cryo-electron microscopy of vitreous sections in the range of nanometer resolution. Thus, unspoiled by chemical fixation, dehydration and contrasting agents, a close to native structure is revealed. Our approach uses perfluorocarbon to substitute the air in the alveoli. Lung tissue was subsequently high-pressure frozen, cryosectioned and observed in a cryo-electron microscope. The lamellar bodies clearly show a tight lamellar morphology. The periodicity of these lamellae was 7.3 nm. Lamellar bifurcations were observed in our cryosections. The technical approach described in this paper allows the examination of the native cellular ultrastructure of the surfactant system under near in vivo conditions, and therefore opens up prospectives for scrutinizing various theories of lamellar body biogenesis, exocytosis and recycling.

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Acknowledgments

This study has been supported in part by grants from the Swiss National Science Foundation (SNF 3100AO-116417) to M.O. and (SNF 31003A-118394) to D.S. The experiments were performed according to the 3R principle and were approved by the cantonal administration of Bern, Switzerland for animal testing (38/09) to D.V.

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Authors and Affiliations

  1. Institute of Anatomy, University of Bern, Bern, Switzerland

    Dimitri Vanhecke, Gudrun Herrmann, Werner Graber, Therese Hillmann-Marti, Daniel Studer & Matthias Ochs

  2. Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany

    Dimitri Vanhecke

  3. Institute of Anatomy and Cell Biology, Justus-Liebig-University Giessen, Giessen, Germany

    Christian Mühlfeld

  4. Institute of Functional and Applied Anatomy, Hannover Medical School, Hannover, Germany

    Matthias Ochs

Authors
  1. Dimitri Vanhecke
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  2. Gudrun Herrmann
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  3. Werner Graber
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  4. Therese Hillmann-Marti
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  5. Christian Mühlfeld
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  6. Daniel Studer
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  7. Matthias Ochs
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Correspondence to Dimitri Vanhecke.

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D. Vanhecke and G. Herrmann contributed equally to this study.

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Vanhecke, D., Herrmann, G., Graber, W. et al. Lamellar body ultrastructure revisited: high-pressure freezing and cryo-electron microscopy of vitreous sections. Histochem Cell Biol 134, 319–326 (2010). https://doi.org/10.1007/s00418-010-0736-4

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