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

Kirk Czymmek; Abhilash Sawant; Kaija Goodman; Janice Pennington; Pal Pedersen; Mrinalini Hoon; Marisa S Otegui

doi:10.1007/978-1-0716-0767-1_7

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

Methods Mol Biol. 2020:2177:69-81. doi: 10.1007/978-1-0716-0767-1_7.

Authors

Kirk Czymmek¹, Abhilash Sawant^{2

3}, Kaija Goodman^{4

5}, Janice Pennington⁵, Pal Pedersen⁶, Mrinalini Hoon^{2

7}, Marisa S Otegui^{8

9}

Affiliations

¹ Advanced Bioimaging Laboratory, Donald Danforth Plant Science Center, St. Louis, MO, USA.
² Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI, USA.
³ Department of Neuroscience, University of Wisconsin-Madison, Madison, WI, USA.
⁴ Department of Botany, University of Wisconsin-Madison, Madison, WI, USA.
⁵ Laboratory of Cell and Molecular Biology, University of Wisconsin-Madison, Madison, WI, USA.
⁶ Carl Zeiss Microscopy, LLC, White Plains, NY, USA.
⁷ McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, WI, USA.
⁸ Department of Botany, University of Wisconsin-Madison, Madison, WI, USA. otegui@wisc.edu.
⁹ Laboratory of Cell and Molecular Biology, University of Wisconsin-Madison, Madison, WI, USA. otegui@wisc.edu.

PMID: 32632806
DOI: 10.1007/978-1-0716-0767-1_7

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.

Keywords: High-pressure freezing; Serial block-face electron microscopy; Three-dimensional electron microscopy.

Publication types

Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Arabidopsis / ultrastructure*
Freeze Substitution
Microscopy, Electron, Scanning / instrumentation*
Microtomy / instrumentation