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Serial sectioning and electron microscopy of large tissue volumes for 3D analysis and reconstruction: a case study of the calyx of Held

Nature Protocols volume 2, pages 9–22 (2007)Cite this article

Abstract

Serial section electron microscopy is typically applied to investigation of small tissue volumes encompassing subcellular structures. However, in neurobiology, the need to relate subcellular structure to organization of neural circuits can require investigation of large tissue volumes at ultrastructural resolution. Analysis of ultrastructure and three-dimensional reconstruction of even one to a few cells is time consuming, and still does not generate the necessary numbers of observations to form well-grounded insights into biological principles. We describe an assemblage of existing computer-based methods and strategies for graphical analysis of large photographic montages to accomplish the study of multiple neurons through large tissue volumes. Sample preparation, data collection and subsequent analyses can be completed within 3–4 months. These methods generate extremely large data sets that can be mined in future studies of nervous system organization.

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Figure 1: Project flow chart showing estimated times for each step.
Figure 2: Flat embedding and re-embedding of tissue for ultrathin sectioning.
Figure 3: EM montages of ultrathin sections.
Figure 4: Required steps for reconstruction of cells from serial EM.
Figure 5: Quantitative measures of calyces of Held in P4 mice.

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Acknowledgements

This work was supported by NIH/NIDCD (DC005035) and an NIH/NCRR COBRE grant (P20 RR14474) to the Sensory Neuroscience Research Center. We acknowledge Janet Cyr and Guy Perkins for constructive comments, Albert Berrebi for introducing G.S. to electron microscopy and Erika Hartweig for demonstrating serial section techniques.

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Authors and Affiliations

  1. Department of Otolaryngology, PO Box 9303, Health Sciences Center, One Medical Center Drive, West Virginia University School of Medicine, Morgantown, 26506-9303, West Virginia, USA

    Brian K Hoffpauir, Brian A Pope & George A Spirou

  2. Department of Physiology and Pharmacology, PO Box 9303, Health Sciences Center, One Medical Center Drive, West Virginia University School of Medicine, Morgantown, 26506-9303, West Virginia, USA

    George A Spirou

  3. Sensory Neuroscience Research Center, PO Box 9303, Health Sciences Center, One Medical Center Drive, West Virginia University School of Medicine, Morgantown, 26506-9303, West Virginia, USA

    Brian K Hoffpauir, Brian A Pope & George A Spirou

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  1. Brian K Hoffpauir
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Correspondence to George A Spirou.

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Supplementary information

Supplementary Fig. 1

Graphical reconstruction of calyces of Held. (PDF 99 kb)

Supplementary Box. 1

Tissue Preparation for Electron Microscopy. (PDF 104 kb)

Supplementary Box. 2

Grid Preparation (6 hours). (PDF 86 kb)

Supplementary Movie

3D reconstruction of a calyx of Held. (MOV 16098 kb)

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Hoffpauir, B., Pope, B. & Spirou, G. Serial sectioning and electron microscopy of large tissue volumes for 3D analysis and reconstruction: a case study of the calyx of Held. Nat Protoc 2, 9–22 (2007). https://doi.org/10.1038/nprot.2007.9

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