Protocol abstract
Nature Protocols 2, - 9 - 22 (2007)
Published online: 31 January 2007 | doi:10.1038/nprot.2007.9
Subject Categories: Neuroscience | Imaging | Isolation, purification and separation | Cell and developmental biology
Serial sectioning and electron microscopy of large tissue volumes for 3D analysis and reconstruction: a case study of the calyx of Held
Brian K Hoffpauir1,3, Brian A Pope1,3 & George A Spirou1,2,3
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.
- Department of Otolaryngology, PO Box 9303, Health Sciences Center, One Medical Center Drive, West Virginia University School of Medicine, Morgantown, West Virginia 26506-9303, USA.
- Department of Physiology and Pharmacology, PO Box 9303, Health Sciences Center, One Medical Center Drive, West Virginia University School of Medicine, Morgantown, West Virginia 26506-9303, USA.
- Sensory Neuroscience Research Center, PO Box 9303, Health Sciences Center, One Medical Center Drive, West Virginia University School of Medicine, Morgantown, West Virginia 26506-9303, USA.
Correspondence to: George A Spirou1,2,3 e-mail: gspirou@hsc.wvu.edu
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A-Z product listing
- 12-well plates(Fisher Scientific)
- 190 proof ethanol(Aaper)
- 200 proof ethanol(Aaper)
- 6.4
25 400 mm glass knife strips(Electron Microscopy Sciences) - Applicator sticks(Fisher Scientific)
- Automated EM Grid Stainer(Leica)