Nanoimaging pp 253-273 | Cite as

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

  • Masaaki Kuwajima
  • John M. Mendenhall
  • Kristen M. Harris
Part of the Methods in Molecular Biology book series (MIMB, volume 950)


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.

Key words

3D reconstruction Hippocampus Microwave-enhanced processing Neuropil Perfusion Scanning electron microscopy Scanning transmission electron microscopy Serial sections Structural plasticity Ultramicrotomy 



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.


  1. 1.
    Gray EG (1959) Electron microscopy of synaptic contacts on dendrite spines of the cerebral cortex. Nature 183:1592–1593PubMedCrossRefGoogle Scholar
  2. 2.
    Palay SL, Palade GE (1955) The fine structure of neurons. J Biophys Biochem Cytol 1:69–88PubMedCrossRefGoogle Scholar
  3. 3.
    Novikoff PM, Novikoff AB, Quintana N, Hauw JJ (1971) Golgi apparatus, GERL, and lysosomes of neurons in rat dorsal root ganglia, studied by thick section and thin section cytochemistry. J Cell Biol 50:859–886PubMedCrossRefGoogle Scholar
  4. 4.
    Spacek J, Lieberman AR (1974) Ultrastructure and three-dimensional organization of synaptic glomeruli in rat somatosensory thalamus. J Anat 117:487–516PubMedGoogle Scholar
  5. 5.
    Thaemert JC (1966) Ultrastructural interrelationships of nerve processes and smooth muscle cells in three dimensions. J Cell Biol 28:37–49PubMedCrossRefGoogle Scholar
  6. 6.
    Williams RC, Kallman F (1955) Interpretations of electron micrographs of single and serial sections. J Biophys Biochem Cytol 1:301–314PubMedCrossRefGoogle Scholar
  7. 7.
    Stevens JK, Davis TL, Friedman N, Sterling P (1980) A systematic approach to reconstructing microcircuitry by electron microscopy of serial sections. Brain Res 2:265–293PubMedCrossRefGoogle Scholar
  8. 8.
    Bock DD, Lee WCA, Kerlin AM et al (2011) Network anatomy and in vivo physiology of visual cortical neurons. Nature 471:177–182PubMedCrossRefGoogle Scholar
  9. 9.
    Briggman KL, Helmstaedter M, Denk W (2011) Wiring specificity in the direction-selectivity circuit of the retina. Nature 471:183–188PubMedCrossRefGoogle Scholar
  10. 10.
    Anderson JR, Jones BW, Watt CB et al (2011) Exploring the retinal connectome. Mol Vis 17:355–379PubMedGoogle Scholar
  11. 11.
    Helmstaedter M, Briggman KL, Denk W (2008) 3D structural imaging of the brain with photons and electrons. Curr Opin Neurobiol 18:633–641PubMedCrossRefGoogle Scholar
  12. 12.
    Mishchenko Y, Hu T, Spacek J, Mendenhall J, Harris KM, Chklovskii DB (2010) Ultrastructural analysis of hippocampal neuropil from the connectomics perspective. Neuron 67:1009–1020PubMedCrossRefGoogle Scholar
  13. 13.
    Bourne JN, Harris KM (2011) Coordination of size and number of excitatory and inhibitory synapses results in a balanced structural plasticity along mature hippocampal CA1 dendrites during LTP. Hippocampus 21:354–373PubMedCrossRefGoogle Scholar
  14. 14.
    Knott GW, Holtmaat A, Wilbrecht L, Welker E, Svoboda K (2006) Spine growth precedes synapse formation in the adult neocortex in vivo. Nat Neurosci 9:1117–1124PubMedCrossRefGoogle Scholar
  15. 15.
    Ostroff LE, Fiala JC, Allwardt B, Harris KM (2002) Polyribosomes redistribute from dendritic shafts into spines with enlarged synapses during LTP in developing rat hippocampal slices. Neuron 35:535–545PubMedCrossRefGoogle Scholar
  16. 16.
    Cantoni M, Knott G, Hébert C (2010) FIB-SEM nanotomography in materials and life science at EPFL. Microsc Microanal 16:226–227CrossRefGoogle Scholar
  17. 17.
    Cantoni M, Genoud C, Hébert C, Knott G (2010) Large volume, isotropic, 3D imaging of cell structure on the nanometer scale. Microsc Anal 24:13–16Google Scholar
  18. 18.
    Knott G, Marchman H, Wall D, Lich B (2008) Serial section scanning electron microscopy of adult brain tissue using focused ion beam milling. J Neurosci 28:2959–2964PubMedCrossRefGoogle Scholar
  19. 19.
    Knott G, Rosset SP, Cantoni M (2011) Focussed ion beam milling and scanning electron microscopy of brain tissue. J Vis Exp 53:e2588. DOI: 10.3791/2588Google Scholar
  20. 20.
    Denk W, Horstmann H (2004) Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol 2:e329PubMedCrossRefGoogle Scholar
  21. 21.
    Hayworth KJ, Kasthuri N, Schalek R, Lichtman JW (2006) Automating the collection of ultrathin serial sections for large volume TEM reconstructions. Microsc Microanal 12:86–87CrossRefGoogle Scholar
  22. 22.
    Micheva KD, Smith SJ (2007) Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron 55:25–36PubMedCrossRefGoogle Scholar
  23. 23.
    Mendenhall JM, Yorston J, Lagarec KG, Bowden J, Harris KM (2009) Large volume high resolution imaging of brain neuropil using SEM-based scanning electron microscopy. Program No. 484.17. 2009 Neuroscience Meeting Planner. Society for Neuroscience, Chicago, IL (Online)Google Scholar
  24. 24.
    Anderson JR, Mohammed S, Grimm B et al (2011) The Viking viewer for connectomics: scalable multi-user annotation and summarization of large volume data sets. J Microsc 241:13–28PubMedCrossRefGoogle Scholar
  25. 25.
    Chklovskii DB, Vitaladevuni S, Scheffer LK (2010) Semi-automated reconstruction of neural circuits using electron microscopy. Curr Opin Neurobiol 20:667–675PubMedCrossRefGoogle Scholar
  26. 26.
    Fiala JC (2005) Reconstruct: a free editor for serial section microscopy. J Microsc 218:52–61PubMedCrossRefGoogle Scholar
  27. 27.
    Helmstaedter M, Briggman KL, Denk W (2011) High-accuracy neurite reconstruction for high-throughput neuroanatomy. Nat Neurosci 14:1081–1088PubMedCrossRefGoogle Scholar
  28. 28.
    Jain V, Seung HS, Turaga SC (2010) Machines that learn to segment images: a crucial technology for connectomics. Curr Opin Neurobiol 20:653–666PubMedCrossRefGoogle Scholar
  29. 29.
    Knowles-Barley S, Butcher NJ, Meinertzhagen IA, Armstrong JD (2011) Biologically inspired EM image alignment and neural reconstruction. Bioinformatics 27:2216–2223PubMedCrossRefGoogle Scholar
  30. 30.
    Lang S, Drouvelis P, Tafaj E, Bastian P, Sakmann B (2011) Fast extraction of neuron morphologies from large-scale SBFSEM image stacks. J Comput Neurosci 31(3):533–545PubMedCrossRefGoogle Scholar
  31. 31.
    Morales J, Alonso-Nanclares L, Rodriguez JR et al (2011) Espina: a tool for the automated segmentation and counting of synapses in large stacks of electron microscopy images. Front Neuroanat 5:18PubMedCrossRefGoogle Scholar
  32. 32.
    Saalfeld S, Cardona A, Hartenstein V, Tomancak P (2010) As-rigid-as-possible mosaicking and serial section registration of large ssTEM datasets. Bioinformatics 26:i57–i63PubMedCrossRefGoogle Scholar
  33. 33.
    Tao-Cheng JH, Gallant PE, Brightman MW, Dosemeci A, Reese TS (2007) Structural changes at synapses after delayed perfusion fixation in different regions of the mouse brain. J Comp Neurol 501:731–740PubMedCrossRefGoogle Scholar
  34. 34.
    Feinberg MD, Szumowski KM, Harris KM (2001) Microwave fixation of rat hippocampal slices. In: Giberson RT, DeMaree RSJ (eds) Microwave techniques and protocols. Humana Press, Totowa, pp 75–88CrossRefGoogle Scholar
  35. 35.
    Harris KM, Perry E, Bourne J, Feinberg M, Ostroff L, Hurlburt J (2006) Uniform serial sectioning for transmission electron microscopy. J Neurosci 26:12101–12103PubMedCrossRefGoogle Scholar
  36. 36.
    Reynolds ES (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol 17:208–212PubMedCrossRefGoogle Scholar
  37. 37.
    FialaJ C, Harris KM (2001) Cylindrical diameters method for calibrating section thickness in serial electron microscopy. J Microsc 202:468–472CrossRefGoogle Scholar
  38. 38.
    Jensen FE, Harris KM (1989) Preservation of neuronal ultrastructure in hippocampal slices using rapid microwave-enhanced fixation. J Neurosci Methods 29:217–230PubMedCrossRefGoogle Scholar
  39. 39.
    Luft JH (1961) Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol 9:409–414PubMedCrossRefGoogle Scholar
  40. 40.
    Kubota Y, Hatada S, Kawaguchi Y (2009) Important factors for the three-dimensional reconstruction of neuronal structures from serial ultrathin sections. Front Neural Circuits 3:4PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Masaaki Kuwajima
    • 1
  • John M. Mendenhall
    • 1
  • Kristen M. Harris
    • 1
    • 2
  1. 1.Center for Learning and MemoryThe University of Texas at AustinAustinUSA
  2. 2.Section of NeurobiologyThe University of Texas at AustinAustinUSA

Personalised recommendations