Imaging Plant Nuclei and Membrane-Associated Cytoskeleton by Field Emission Scanning Electron Microscopy - Plant Cell Morphogenesis: Methods and Protocols - page 166

Chapter 14

Imaging Plant Nuclei and Membrane-Associated
Cytoskeleton by Field Emission Scanning Electron
Microscopy

Jindřiška Fišerová and Martin W. Goldberg

Abstract

Scanning electron microscopy (SEM) is a powerful technique that can image exposed surfaces in 3D.
Modern scanning electron microscopes, with fi eld emission electron sources and in-lens specimen cham-
bers, achieve resolutions of better than 0.5 nm and thus offer views of ultrastructural details of subcellular
structures or even macromolecular complexes. Obtaining a reliable image is, however, dependent on sam-
ple preparation methods that robustly but accurately preserve biological structures. In plants, exposing the
object of interest may be diffi cult due to the existence of a cell wall. This protocol shows how to isolate
plant nuclei for SEM imaging of the nuclear envelope and associated structures from both sides of the
nuclear envelope in cultured cells as well as in leaf or root cells. Further, it provides a method for uncover-
ing membrane-associated cytoskeletal structures.

Key words Scanning electron microscopy, Plant, SEM, Tobacco, Critical point drying, Coating,
Wet fracture, Nucleus, Nuclear envelope, Cytoskeleton

1 Introduction

Scanning electron microscopy (SEM) provides useful 3D informa-
tion about structural details of surfaces of tissues, cells, organelles,
or even single proteins. Unlike light microscopy, which can take
advantage of in vivo detection of cells, cellular movements, or
movements of individual proteins, the sample to be observed under
a conventional SEM must be fi xed, dehydrated, and metal coated
fi rst. Thus, the sample is subjected to signifi cant manipulation, and
damage to the structures of interest may occur. Therefore, it is