Chapter 5
Progress Towards CryoEM: Negative-Stain Procedures
for Biological Samples
Shane Gonen
Abstract
In recent years, electron cryo-microscopy (CryoEM) has become a powerful method for the highresolution studies of biological macromolecules. While CryoEM experiments can begin without additional
microscopy steps, negative-stain EM can tremendously minimize CryoEM screening. Negative-stain is a
quick method that can be used to screen for robust biochemical conditions, the integrity, binding, and
composition of samples and to get an estimation of sample grid concentration. For some applications, the
map resolutions potentially afforded by stain may be as biologically informative as in CryoEM. Here, I
describe the benefits and pitfalls of negative-stain EM, with particular emphasis on Uranyl stains with the
main goal of screening in advance of CryoEM. In addition, I provide a materials list, detailed protocol and
possible adjustments for the use of stains for biological samples requiring imaging and/or diffraction-based
methods of EM.
Key words Negative-stain, Uranyl formate, Uranyl acetate, CryoEM, Single-particle, Averaging,
Electron crystallography, Crystals, Electron microscopy
1 Introduction
Electron cryo-microscopy (CryoEM) has gained tremendous
momentum over the last few years with the advent of new microscopes with stable optics, more sensitive and fast cameras capable of
taking movies and faster, more powerful and accessible software for
data collection, correcting motion and processing [1–5]. Traditionally, a typical CryoEM workflow first involved screening using
negative-stain electron microscopy (EM), which is a very quick,
albeit low-resolution method to visualize biological samples [6]
(Figs. 1, 2, and 3). Due to the powerful nature of modern EM,
negative-stain is sometimes skipped in favor of direct CryoEM
workflows. For some samples, such as very stable macromolecules
[7] or those where precious little amount is available (and highresolution paramount), directly going to CryoEM is likely a better
choice. However, for many samples, especially those that can be
Tamir Gonen and Brent L. Nannenga (eds.), CryoEM: Methods and Protocols, Methods in Molecular Biology, vol. 2215,
https://doi.org/10.1007/978-1-0716-0966-8_5, © Springer Science+Business Media, LLC, part of Springer Nature 2021
115
Progress Towards CryoEM: Negative-Stain Procedures
for Biological Samples
Shane Gonen
Abstract
In recent years, electron cryo-microscopy (CryoEM) has become a powerful method for the highresolution studies of biological macromolecules. While CryoEM experiments can begin without additional
microscopy steps, negative-stain EM can tremendously minimize CryoEM screening. Negative-stain is a
quick method that can be used to screen for robust biochemical conditions, the integrity, binding, and
composition of samples and to get an estimation of sample grid concentration. For some applications, the
map resolutions potentially afforded by stain may be as biologically informative as in CryoEM. Here, I
describe the benefits and pitfalls of negative-stain EM, with particular emphasis on Uranyl stains with the
main goal of screening in advance of CryoEM. In addition, I provide a materials list, detailed protocol and
possible adjustments for the use of stains for biological samples requiring imaging and/or diffraction-based
methods of EM.
Key words Negative-stain, Uranyl formate, Uranyl acetate, CryoEM, Single-particle, Averaging,
Electron crystallography, Crystals, Electron microscopy
1 Introduction
Electron cryo-microscopy (CryoEM) has gained tremendous
momentum over the last few years with the advent of new microscopes with stable optics, more sensitive and fast cameras capable of
taking movies and faster, more powerful and accessible software for
data collection, correcting motion and processing [1–5]. Traditionally, a typical CryoEM workflow first involved screening using
negative-stain electron microscopy (EM), which is a very quick,
albeit low-resolution method to visualize biological samples [6]
(Figs. 1, 2, and 3). Due to the powerful nature of modern EM,
negative-stain is sometimes skipped in favor of direct CryoEM
workflows. For some samples, such as very stable macromolecules
[7] or those where precious little amount is available (and highresolution paramount), directly going to CryoEM is likely a better
choice. However, for many samples, especially those that can be
Tamir Gonen and Brent L. Nannenga (eds.), CryoEM: Methods and Protocols, Methods in Molecular Biology, vol. 2215,
https://doi.org/10.1007/978-1-0716-0966-8_5, © Springer Science+Business Media, LLC, part of Springer Nature 2021
115