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Volume Microscopy pp 121–149Cite as

Correlative Ultrastructural Analysis of Functionally Modulated Synapses Using Automated Tape-Collecting Ultramicrotome and SEM Array Tomography

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Part of the book series: Neuromethods ((NM,volume 155))

Abstract

Live imaging of dendritic spines using advanced light microscopy (LM) provides insight into how the brain processes information to learn and form memories. As a complementary approach, electron microscopy (EM) offers a complete view of the ultrastructural characteristics of synapses, such as the size of postsynaptic density, as well as the distribution and number of synaptic vesicles in the presynaptic terminal. By bridging these two different visualization platforms, function and ultrastructure can be directly linked at the level of individual synapses. The technical challenge is how to examine the same spines in reliable and reproducible ways using two imaging modalities with completely different spatial scales. Here, we describe our detailed workflow to combine light and electron microscopy for efficient correlative analysis of spines of interest. As an example, we show how to find a dendritic spine that is stimulated with 2-photon glutamate uncaging on a CA1 pyramidal neuron expressing green fluorescent protein (GFP) in organotypic hippocampal slices. Following fluorescence observation under a 2-photon fluorescence microscope, the tissue is processed for EM using pre-embedding immunogold-labeling of GFP to locate the cell of interest. It is then sectioned with the Automated Tape Collecting Ultramicrotome (ATUMtome) to reliably and quickly collect hundreds of serial sections from a large block face (up to 3 × 3 mm). Then using a scanning electron microscope (SEM) in combination with array tomography software (Atlas 5 AT), we semiautomatically collect images at multiple resolutions. The obtained volumetric dataset is reconstructed and analyzed in a 3D manner. This workflow allows us to collect data for quantitative analysis faster than conventional serial sectioning followed by transmission electron microscopy (TEM) imaging.

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Acknowledgments

The authors thank RMC Boeckeler and Carl Zeiss Microscopy for their support during startup and to keep the equipment up and running to establish the workflow. This work was supported by funding from Max Planck Society and a grant from Florida Atlantic University Pilot Graduate Research and Inquiry Program (GRIP).

Author information

Authors and Affiliations

  1. Neural Signal Transduction Research Group, Max Planck Florida Institute for Neuroscience, Jupiter, FL, USA

    Ye Sun, Takayasu Mikuni & Ryohei Yasuda

  2. Integrative Biology and Neuroscience Graduate Program, Florida Atlantic University, Jupiter, FL, USA

    Ye Sun, Ryohei Yasuda & Naomi Kamasawa

  3. International Max Planck Research School for Brain and Behavior, Florida Atlantic University, Jupiter, FL, USA

    Ye Sun & Ryohei Yasuda

  4. Imaging Center and Electron Microscopy Facility, Max Planck Florida Institute for Neuroscience, Jupiter, FL, USA

    Connon Thomas, Debbie Guerrero-Given & Naomi Kamasawa

Authors
  1. Ye Sun
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  2. Connon Thomas
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  3. Takayasu Mikuni
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  4. Debbie Guerrero-Given
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  5. Ryohei Yasuda
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  6. Naomi Kamasawa
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Corresponding author

Correspondence to Naomi Kamasawa .

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Editors and Affiliations

  1. Cryo EM, Centre for Advanced Materials (CAM), Universität Heidelberg, Heidelberg, Germany

    Irene Wacker

  2. Sector Life Science, Carl Zeiss Microscopy GmbH, Oberkochen, Germany, Carl Zeiss, Oberkochen, Germany

    Eric Hummel

  3. BioQuant, Universität Heidelberg, Heidelberg, Germany, Cryo EM, BioQuant, Universitätsklinikum Heidelberg, Heidelberg, Germany

    Steffen Burgold

  4. Cryo EM, Centre for Advanced Materials (CAM), Universität Heidelberg, Heidelberg, Germany, Cryo EM, BioQuant, Universitätsklinikum Heidelberg, Heidelberg, Germany

    Rasmus Schröder

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Sun, Y., Thomas, C., Mikuni, T., Guerrero-Given, D., Yasuda, R., Kamasawa, N. (2020). Correlative Ultrastructural Analysis of Functionally Modulated Synapses Using Automated Tape-Collecting Ultramicrotome and SEM Array Tomography. In: Wacker, I., Hummel, E., Burgold, S., Schröder, R. (eds) Volume Microscopy . Neuromethods, vol 155. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0691-9_7

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  • DOI: https://doi.org/10.1007/978-1-0716-0691-9_7

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