sample is challenging. Over the years, many obstacles were overcome to reveal biomolecules in atomic details.
For most biological applications, the sample is applied onto
3 mm EM grids that can be made of different material. Continuously carbon-coated grids are used for negative staining where
carbon acts as a support for the sample, whereas perforated
carbon-coated grids can in addition be used in cryo-EM when
adsorption has to be avoided. The carbon surface is generally
hydrophobic and has to be rendered hydrophilic to allow proper
spreading of the sample and adsorption of the biomolecules
[2]. The properties of the carbon surface are altered by glow
discharge, a process where the grids are exposed to an ionizing
plasma formed by applying an electric current through a gas volume
at low pressure. The ionized gas molecules will modify the properties of the carbon surface of the grids and render it hydrophilic.
We will describe two popular specimen preparation protocols
for subsequent single-particle electron microscopy observation,
namely negative staining and vitrification.
1.1 Negative
Staining
The most straightforward negative staining approach was first
described by Brenner and Horne in 1959 [3]. This method brings
important preliminary information on sample quality and may be
used on a daily basis to follow and optimize a purification protocol.
Associated with single particle image analysis, it may provide important information on homogeneity or oligomerization states. This
information may not be portable for cryo-EM sample preparation
but guaranties a firm starting point. Negative staining consists of
embedding the protein into a matrix of heavy atom salt to increase
the contrast of unstained biomolecules and prevent, to some
extent, their collapse during the drying process when exposed to
the vacuum of the microscope. Stains of heavy metal salts, such as
uranyl acetate, uranyl formate, phosphotungstic acid, and others,
are commonly used, since they strongly scatter electrons and produce high amplitude contrast. It is important to notice that negative staining reveals only the surface and the overall shape of protein
molecules and does not provide information about their inner
structure. The resolution that can be achieved after image analysis
of multiple images is limited to 15–20 A ˚ , by the graininess of the
stain and particle distortions during the drying process. This
method is, however, very useful to check particle homogeneity
and to determine initial 3D models. Furthermore, the sample
preparation is easy to perform and to observe at room temperature
in a standard transmission electron microscope. The stained sample, or more specifically, its heavy metal cast, is more resistant to
radiation damage produced by the electron irradiation, and the
high image contrast favors image interpretation.
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Alexandre Frechard et al.
For most biological applications, the sample is applied onto
3 mm EM grids that can be made of different material. Continuously carbon-coated grids are used for negative staining where
carbon acts as a support for the sample, whereas perforated
carbon-coated grids can in addition be used in cryo-EM when
adsorption has to be avoided. The carbon surface is generally
hydrophobic and has to be rendered hydrophilic to allow proper
spreading of the sample and adsorption of the biomolecules
[2]. The properties of the carbon surface are altered by glow
discharge, a process where the grids are exposed to an ionizing
plasma formed by applying an electric current through a gas volume
at low pressure. The ionized gas molecules will modify the properties of the carbon surface of the grids and render it hydrophilic.
We will describe two popular specimen preparation protocols
for subsequent single-particle electron microscopy observation,
namely negative staining and vitrification.
1.1 Negative
Staining
The most straightforward negative staining approach was first
described by Brenner and Horne in 1959 [3]. This method brings
important preliminary information on sample quality and may be
used on a daily basis to follow and optimize a purification protocol.
Associated with single particle image analysis, it may provide important information on homogeneity or oligomerization states. This
information may not be portable for cryo-EM sample preparation
but guaranties a firm starting point. Negative staining consists of
embedding the protein into a matrix of heavy atom salt to increase
the contrast of unstained biomolecules and prevent, to some
extent, their collapse during the drying process when exposed to
the vacuum of the microscope. Stains of heavy metal salts, such as
uranyl acetate, uranyl formate, phosphotungstic acid, and others,
are commonly used, since they strongly scatter electrons and produce high amplitude contrast. It is important to notice that negative staining reveals only the surface and the overall shape of protein
molecules and does not provide information about their inner
structure. The resolution that can be achieved after image analysis
of multiple images is limited to 15–20 A ˚ , by the graininess of the
stain and particle distortions during the drying process. This
method is, however, very useful to check particle homogeneity
and to determine initial 3D models. Furthermore, the sample
preparation is easy to perform and to observe at room temperature
in a standard transmission electron microscope. The stained sample, or more specifically, its heavy metal cast, is more resistant to
radiation damage produced by the electron irradiation, and the
high image contrast favors image interpretation.
244
Alexandre Frechard et al.