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Large-Volume Reconstruction of Brain Tissue from High-Resolution Serial Section Images Acquired by SEM-Based Scanning Transmission Electron Microscopy

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Nanoimaging

Part of the book series: Methods in Molecular Biology ((MIMB,volume 950))

Abstract

With recent improvements in instrumentation and computational tools, serial section electron microscopy has become increasingly straightforward. A new method for imaging ultrathin serial sections is developed based on a field emission scanning electron microscope fitted with a transmitted electron detector. This method is capable of automatically acquiring high-resolution serial images with a large field size and very little optical and physical distortions. In this chapter, we describe the procedures leading to the generation and analyses of a large-volume stack of high-resolution images (64 μm × 64 μm × 10 μm, or larger, at 2 nm pixel size), including how to obtain large-area serial sections of uniform thickness from well-preserved brain tissue that is rapidly perfusion-fixed with mixed aldehydes, processed with a microwave-enhanced method, and embedded into epoxy resin.

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Acknowledgment

We thank Drs. Cliff Abraham and Jared Bowden for tissue samples from which images in the Figs. 2, 3, and 4 were taken. We also thank Laurence Lindsey for help with computational tools and Patrick Parker for help in preparing the manuscript. This work was supported by the US National Institutes of Health grants NS021184, NS033574, and EB002170 to K.M.H. and the Texas Emerging Technology Fund.

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Authors and Affiliations

  1. Center for Learning and Memory, The University of Texas at Austin, Austin, TX, USA

    Masaaki Kuwajima, John M. Mendenhall & Kristen M. Harris

  2. Section of Neurobiology, The University of Texas at Austin, Austin, TX, USA

    Kristen M. Harris

Authors
  1. Masaaki Kuwajima
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  2. John M. Mendenhall
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  3. Kristen M. Harris
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Corresponding author

Correspondence to Kristen M. Harris .

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Editors and Affiliations

  1. Nat. Inst. Biomedical Imaging & Bioengin, National Institutes of Health, South Drive 13, Bethesda, 20892, Maryland, USA

    Alioscka A. Sousa

  2. , National Cancer Institute, National Institutes of Health, Center Drive 10, Bethesda, 20892, USA

    Michael J. Kruhlak

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Kuwajima, M., Mendenhall, J.M., Harris, K.M. (2013). Large-Volume Reconstruction of Brain Tissue from High-Resolution Serial Section Images Acquired by SEM-Based Scanning Transmission Electron Microscopy. In: Sousa, A., Kruhlak, M. (eds) Nanoimaging. Methods in Molecular Biology, vol 950. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-137-0_15

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  • DOI: https://doi.org/10.1007/978-1-62703-137-0_15

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-136-3

  • Online ISBN: 978-1-62703-137-0

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