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Imaging Plant Cells by High-Pressure Freezing and Serial block-face scanning electron microscopy

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Plant Endosomes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2177))

Abstract

This chapter describes methods to enhanced contrast of plant material processed by high-pressure freezing and freeze substitution for improved visualization by serial block-face scanning electron microscopy (SBEM). The contrast enhancing steps are based on a protocol involving the sequential incubation of samples in heavy metals and sodium thiocarbohydrazide (OTO staining). We also describe the pipeline for imaging plant tissues in a commercial SBEM system (Gatan 3View®) and routines for the image analysis and three-dimensional reconstructions using open-source and commercial software packages.

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Acknowledgments

This work was partially funded by NSF grant MCB1614965 to MSO and UW2020 WARF Discovery award to M.H.

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Authors and Affiliations

  1. Advanced Bioimaging Laboratory, Donald Danforth Plant Science Center, St. Louis, MO, USA

    Kirk Czymmek

  2. Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI, USA

    Abhilash Sawant & Mrinalini Hoon

  3. Department of Neuroscience, University of Wisconsin-Madison, Madison, WI, USA

    Abhilash Sawant

  4. Department of Botany, University of Wisconsin-Madison, Madison, WI, USA

    Kaija Goodman & Marisa S. Otegui

  5. Laboratory of Cell and Molecular Biology, University of Wisconsin-Madison, Madison, WI, USA

    Kaija Goodman, Janice Pennington & Marisa S. Otegui

  6. Carl Zeiss Microscopy, LLC, White Plains, NY, USA

    Pal Pedersen

  7. McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, WI, USA

    Mrinalini Hoon

Authors
  1. Kirk Czymmek
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  2. Abhilash Sawant
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  3. Kaija Goodman
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  4. Janice Pennington
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  5. Pal Pedersen
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  6. Mrinalini Hoon
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  7. Marisa S. Otegui
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Corresponding author

Correspondence to Marisa S. Otegui .

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Editors and Affiliations

  1. Department of Botany and Laboratory of Cell and Molecular Biology, University of Wisconsin-Madison, Madison, WI, USA

    Marisa S. Otegui

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Czymmek, K. et al. (2020). Imaging Plant Cells by High-Pressure Freezing and Serial block-face scanning electron microscopy. In: Otegui, M. (eds) Plant Endosomes. Methods in Molecular Biology, vol 2177. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0767-1_7

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  • DOI: https://doi.org/10.1007/978-1-0716-0767-1_7

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0766-4

  • Online ISBN: 978-1-0716-0767-1

  • eBook Packages: Springer Protocols

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