1.2 Vitrification
The major drawback of negative staining is that the sample is dried
during preparation and introduction into the microscope vacuum.
To overcome this limitation, several options have been explored to
keep the specimen hydrated including the construction of hydrated
chambers around the specimen holder. Attempts were made to
replace water with sugar polymers to functionally replace the shielding properties of water, and remarkable results were obtained for
2D crystals of membrane proteins. Cryogenic methods were tried
early on and proved efficient to reduce radiation damage resulting
from the interaction of electrons with matter by a factor of 2 at
liquid nitrogen temperature. However, freezing water invariably
resulted in ice crystal formation which affects sample distribution
and image quality. Being able to obtain vitreous ice at atmospheric
pressure was a major breakthrough for studying biological macromolecules in a fully hydrated state in the electron microscope. The
method consists of flash freezing a thin layer of an aqueous suspension of the purified biomolecule to form a vitrified sample that can
be transferred and observed in a cryo electron microscope at liquid
nitrogen temperature. A layer of suspension thinner than 100 nm is
generally obtained by removing the excess of a drop of sample
applied onto the electron microscopy grid by blotting with a filter
paper. Alternatively, sample micro droplets can be sprayed onto the
grid or applied by a nanoliter dispensers [4]. Vitrification is generally performed by plunging the EM grid into liquid ethane cooled
by liquid nitrogen for better heat dissipation. High pressure freezing devices have been developed for the vitrification of up to
300 nm thick sample [5]. This approach, which was rewarded the
Nobel Prize in chemistry in 2017, preserves the hydrated structure
of the molecules at atomic resolution. The vitrification process can
be performed once the sample is adsorbed on a carbon film, but
adsorption can be avoided by using holey carbon films. In this case,
observations are made on the thin layer of vitrified suspension
stretched over the carbon-free holes. However, the sample still
experiences the interaction with the air–water interface which can
be deleterious when the specimen is prone to denaturation or can
result in preferential orientations of the molecules [6].
2 Materials
We list here the equipment needed to prepare purified biological
molecules for their observation in electron microscopy. The negative staining and vitrification methods require common material
listed in Subheading 2.1 and have their own specificities detailed
in Subheadings 2.2 and 2.3, respectively.
Specimen Optimization for Single Particle cryo-EM
245
The major drawback of negative staining is that the sample is dried
during preparation and introduction into the microscope vacuum.
To overcome this limitation, several options have been explored to
keep the specimen hydrated including the construction of hydrated
chambers around the specimen holder. Attempts were made to
replace water with sugar polymers to functionally replace the shielding properties of water, and remarkable results were obtained for
2D crystals of membrane proteins. Cryogenic methods were tried
early on and proved efficient to reduce radiation damage resulting
from the interaction of electrons with matter by a factor of 2 at
liquid nitrogen temperature. However, freezing water invariably
resulted in ice crystal formation which affects sample distribution
and image quality. Being able to obtain vitreous ice at atmospheric
pressure was a major breakthrough for studying biological macromolecules in a fully hydrated state in the electron microscope. The
method consists of flash freezing a thin layer of an aqueous suspension of the purified biomolecule to form a vitrified sample that can
be transferred and observed in a cryo electron microscope at liquid
nitrogen temperature. A layer of suspension thinner than 100 nm is
generally obtained by removing the excess of a drop of sample
applied onto the electron microscopy grid by blotting with a filter
paper. Alternatively, sample micro droplets can be sprayed onto the
grid or applied by a nanoliter dispensers [4]. Vitrification is generally performed by plunging the EM grid into liquid ethane cooled
by liquid nitrogen for better heat dissipation. High pressure freezing devices have been developed for the vitrification of up to
300 nm thick sample [5]. This approach, which was rewarded the
Nobel Prize in chemistry in 2017, preserves the hydrated structure
of the molecules at atomic resolution. The vitrification process can
be performed once the sample is adsorbed on a carbon film, but
adsorption can be avoided by using holey carbon films. In this case,
observations are made on the thin layer of vitrified suspension
stretched over the carbon-free holes. However, the sample still
experiences the interaction with the air–water interface which can
be deleterious when the specimen is prone to denaturation or can
result in preferential orientations of the molecules [6].
2 Materials
We list here the equipment needed to prepare purified biological
molecules for their observation in electron microscopy. The negative staining and vitrification methods require common material
listed in Subheading 2.1 and have their own specificities detailed
in Subheadings 2.2 and 2.3, respectively.
Specimen Optimization for Single Particle cryo-EM
245