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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
Brian K Hoffpauir1,3, Brian A Pope1,3, George A Spirou13
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1Department of Otolaryngology, 2Department of Physiology and Pharmacology, 3Sensory 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 should be addressed to G.A.S. ([email protected]).
Published online 31 January 2007; doi:10.1038/nprot.2007.9
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 34 months. These methods generate extremely large data sets that can be mined in future studies of nervous system organization.
: INTRODUCTIONIndividual neurons inuence and are inuenced by the neural circuits in which they reside. It is increasingly clear that the analysis of events at a subcellular or cellular scale is best interpreted when placed in the setting of larger dimensions of neural interactions.Experimental techniques are typically designed for particular scales of analyses, and only with great effort are they applied to larger scales of tissue organization. A good example is recording from single units, which only through persistence generates data from a sufciently large population of cells. Advantages in technology, such as arrays of recording electrodes1,2, permit simultaneous
recording of multiple single units, but challenges remain in collecting sufcient numbers of quality recordings and then manipulating and analyzing these large data sets.
Investigating the structural underpinnings of cell function also requires analytical approaches from...