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Ultrastructural Observation of Glutamatergic Synapses by Focused Ion Beam Scanning Electron Microscopy (FIB/SEM)

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Glutamate Receptors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1941))

Abstract

A thorough understanding of the synaptic ultrastructure is necessary to bridge our current knowledge gap about the relationship between neuronal structure and function. Recent development of focused ion beam scanning electron microscopy (FIB/SEM) has made it possible to image neuronal structures with high speed and efficiency. Here, we present our routine protocol for correlative two-photon microscopy and FIB/SEM imaging of glutamatergic synapses. Femtosecond-pulsed near-infrared laser was used to create fiducial marks around the dendrite of interest in aldehyde-fixed tissues. Thereafter, samples were subjected to en bloc staining with rOTO (reduced osmium tetroxide-thiocarbohydrazide-osmium tetroxide), followed by lead aspartate and uranyl acetate to enhance tissue contrast. Reliable detection of postsynaptic density (PSD) and plasma membrane contours by the sample preparation protocol optimized for FIB/SEM allows us to precisely evaluate morphological features that shape glutamatergic synaptic transmission.

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Authors and Affiliations

  1. Department of Cellular Neurobiology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

    Ai Takahashi-Nakazato, Laxmi Kumar Parajuli, Hirohide Iwasaki, Shinji Tanaka & Shigeo Okabe

  2. CREST, JST, Tokyo, Japan

    Ai Takahashi-Nakazato, Laxmi Kumar Parajuli, Hirohide Iwasaki, Shinji Tanaka & Shigeo Okabe

Authors
  1. Ai Takahashi-Nakazato
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  2. Laxmi Kumar Parajuli
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  3. Hirohide Iwasaki
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  4. Shinji Tanaka
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  5. Shigeo Okabe
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Corresponding author

Correspondence to Shigeo Okabe .

Editor information

Editors and Affiliations

  1. Department of Neurology, University of Wisconsin, Madison, WI, USA

    Corinna Burger

  2. ANSER, Homeland Security Studies and Analysis Institute, Falls Church, VA, USA

    Margaret Jo Velardo

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Takahashi-Nakazato, A., Parajuli, L.K., Iwasaki, H., Tanaka, S., Okabe, S. (2019). Ultrastructural Observation of Glutamatergic Synapses by Focused Ion Beam Scanning Electron Microscopy (FIB/SEM). In: Burger, C., Velardo, M. (eds) Glutamate Receptors. Methods in Molecular Biology, vol 1941. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9077-1_2

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  • DOI: https://doi.org/10.1007/978-1-4939-9077-1_2

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9076-4

  • Online ISBN: 978-1-4939-9077-1

  • eBook Packages: Springer Protocols

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