Chapter 13
Optimization of Sample Preparation for the Observation
of Macromolecular Complexes by Electron (cryo-)
Microscopy
Alexandre Frechard, Grigory Sharov, Maximilien Werderer,
and Patrick Schultz
Abstract
Electron microscopy is a powerful tool for studying the homogeneity and structure of biomolecular
complexes. The small wavelength of electron and the availability of electron optics enable the direct
visualization of macromolecular assemblies in a large range of sizes between 5 and 100 nm. This informs
us about the degree of multimerization or aggregation and provides precise information about their general
shape and dimensions. When combined with sophisticated image analysis protocols, three-dimensional
(3D) information can be gained from 2D projections of the sample, leading to a structural description.
When intermediate steps of a reaction can be imaged, insights into the mode of action of macromolecules
can be gained, and structure–function relations can be established. However, the way the sample is prepared
for its observation within the vacuum of an electron microscope determines the information that can be
retrieved from the experiment. We will review two commonly used specimen preparation protocols for
subsequent single-particle electron microscopy observation, namely negative staining and vitrification.
Key words Single particle electron microscopy, Negative stain electron microscopy, Cryo-EM,
EM sample preparation
1 Introduction
In the last decades, imaging of biological macromolecules by electron microscopy became a major structural biology method to
describe the shape and size of macromolecules, to investigate
their oligomeric or aggregated state, and, in combination with
single particle image analysis, to describe their structure [1]. Ultimately this approach aims at understanding the mode of action of
molecular assemblies by correlating their structure with their function. Handling macromolecular assemblies a few nanometers in size
for their observation in the vacuum of a transmission electron
microscope (TEM) while preserving the structural integrity of the
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_13, © Springer Science+Business Media, LLC, part of Springer Nature 2021
243
Optimization of Sample Preparation for the Observation
of Macromolecular Complexes by Electron (cryo-)
Microscopy
Alexandre Frechard, Grigory Sharov, Maximilien Werderer,
and Patrick Schultz
Abstract
Electron microscopy is a powerful tool for studying the homogeneity and structure of biomolecular
complexes. The small wavelength of electron and the availability of electron optics enable the direct
visualization of macromolecular assemblies in a large range of sizes between 5 and 100 nm. This informs
us about the degree of multimerization or aggregation and provides precise information about their general
shape and dimensions. When combined with sophisticated image analysis protocols, three-dimensional
(3D) information can be gained from 2D projections of the sample, leading to a structural description.
When intermediate steps of a reaction can be imaged, insights into the mode of action of macromolecules
can be gained, and structure–function relations can be established. However, the way the sample is prepared
for its observation within the vacuum of an electron microscope determines the information that can be
retrieved from the experiment. We will review two commonly used specimen preparation protocols for
subsequent single-particle electron microscopy observation, namely negative staining and vitrification.
Key words Single particle electron microscopy, Negative stain electron microscopy, Cryo-EM,
EM sample preparation
1 Introduction
In the last decades, imaging of biological macromolecules by electron microscopy became a major structural biology method to
describe the shape and size of macromolecules, to investigate
their oligomeric or aggregated state, and, in combination with
single particle image analysis, to describe their structure [1]. Ultimately this approach aims at understanding the mode of action of
molecular assemblies by correlating their structure with their function. Handling macromolecular assemblies a few nanometers in size
for their observation in the vacuum of a transmission electron
microscope (TEM) while preserving the structural integrity of the
Arnaud Poterszman (ed.), Multiprotein Complexes: Methods and Protocols, Methods in Molecular Biology, vol. 2247,
https://doi.org/10.1007/978-1-0716-1126-5_13, © Springer Science+Business Media, LLC, part of Springer Nature 2021
243