process in the late 1970s and published the work in the year 1981 [11]. They
showed that sample in buffer/water must be cooled in less than a millisecond to
avoid the ice crystal formation and to get amorphous ice (i.e., vitrified). They also
showed that if the temperature of specimen is kept sufficiently low below −160 °C,
the vitrified state could be maintained for long time [11, 23]. This seminal discovery
enabled proteins to be visualized in its native state under the vacuum of transmission electron microscope (TEM). For this discovery, Dubochet received
one-third of the Noble Prize in Chemistry in the year 2017. The solution-state
protein sample is frozen in time and space, maintaining the integrity of the protein’s
structural state in the vitrified water. The vitrification can be carried out with a
homemade manual plunger or using a commercially available vitrification robot.
A perfect vitrified specimen is one in which the thickness of the ice over the holes
of the grid is such that there is one single layer of particles distributed, the particles
are uniformly distributed (with distance between each particles at least 1.5 times the
particle size), and the particles adopt as many different orientations as possible. The
vitrified specimen grid is then placed in a cryo grid storage box that is preserved in
liquid nitrogen storage Dewar, until the data collection is carried on a
high-resolution cryo-TEM. An extensive description of the specimen preparation is
given in Passmore and Russo [24].
2.2 Data Collection
Data collection is carried out on a cryo-TEM equipped with a 200 or 300 kV field
emission gun (FEG) necessary to obtain a high-resolution single-particle data. The
stored grids are transferred from the cryo grid storage box to a single tilt
cryo-transfer holder pre-cooled on a cryo-workstation (Fig. 2b). In this case, only
one grid can be inserted into the TEM by manually loading the holder into the
cryo-TEM (Fig. 2a) and analyzed before the holder is taken out of the microscope
at the end of data collection. Alternatively, each one of the stored grids can be
transferred one by one to a cartridge, which is then placed on multiple grid holder
cassette (which holds up to 12 grids). This cassette is then placed into the capsule,
which is loaded into the cryo-TEM (Fig. 2c) through an autoloader robot that is
built in the microscope. Thermo Fisher Scientific Talos Arctica/Glacios, Thermo
Fisher Scientific Krios, and JEOL Cryo ARM 200/300 are microscopes with such
autoloader capabilities. The robotic grid loader then can load one by one to the
stage using inbuilt robot, which can load or unload the grid on the stage controlled
by software. In case of high-end TEM analysis, grid atlas can be created to choose
the square of right thickness from all the loaded grids. It is very important to keep
the grid always under liquid nitrogen in order to avoid any ice crystal formation and
contamination on the grid. Hence, all the processes described in Fig. 2, which
involve handling of frozen specimen grid, are carried out under liquid nitrogen. Ice
crystals destroy the view of particles by dark contrast, and hence, it is critical to
avoid any exposure of plunge-frozen grid to the air.
Single-Particle cryo-EM as a Pipeline for Obtaining Atomic …
381
showed that sample in buffer/water must be cooled in less than a millisecond to
avoid the ice crystal formation and to get amorphous ice (i.e., vitrified). They also
showed that if the temperature of specimen is kept sufficiently low below −160 °C,
the vitrified state could be maintained for long time [11, 23]. This seminal discovery
enabled proteins to be visualized in its native state under the vacuum of transmission electron microscope (TEM). For this discovery, Dubochet received
one-third of the Noble Prize in Chemistry in the year 2017. The solution-state
protein sample is frozen in time and space, maintaining the integrity of the protein’s
structural state in the vitrified water. The vitrification can be carried out with a
homemade manual plunger or using a commercially available vitrification robot.
A perfect vitrified specimen is one in which the thickness of the ice over the holes
of the grid is such that there is one single layer of particles distributed, the particles
are uniformly distributed (with distance between each particles at least 1.5 times the
particle size), and the particles adopt as many different orientations as possible. The
vitrified specimen grid is then placed in a cryo grid storage box that is preserved in
liquid nitrogen storage Dewar, until the data collection is carried on a
high-resolution cryo-TEM. An extensive description of the specimen preparation is
given in Passmore and Russo [24].
2.2 Data Collection
Data collection is carried out on a cryo-TEM equipped with a 200 or 300 kV field
emission gun (FEG) necessary to obtain a high-resolution single-particle data. The
stored grids are transferred from the cryo grid storage box to a single tilt
cryo-transfer holder pre-cooled on a cryo-workstation (Fig. 2b). In this case, only
one grid can be inserted into the TEM by manually loading the holder into the
cryo-TEM (Fig. 2a) and analyzed before the holder is taken out of the microscope
at the end of data collection. Alternatively, each one of the stored grids can be
transferred one by one to a cartridge, which is then placed on multiple grid holder
cassette (which holds up to 12 grids). This cassette is then placed into the capsule,
which is loaded into the cryo-TEM (Fig. 2c) through an autoloader robot that is
built in the microscope. Thermo Fisher Scientific Talos Arctica/Glacios, Thermo
Fisher Scientific Krios, and JEOL Cryo ARM 200/300 are microscopes with such
autoloader capabilities. The robotic grid loader then can load one by one to the
stage using inbuilt robot, which can load or unload the grid on the stage controlled
by software. In case of high-end TEM analysis, grid atlas can be created to choose
the square of right thickness from all the loaded grids. It is very important to keep
the grid always under liquid nitrogen in order to avoid any ice crystal formation and
contamination on the grid. Hence, all the processes described in Fig. 2, which
involve handling of frozen specimen grid, are carried out under liquid nitrogen. Ice
crystals destroy the view of particles by dark contrast, and hence, it is critical to
avoid any exposure of plunge-frozen grid to the air.
Single-Particle cryo-EM as a Pipeline for Obtaining Atomic …
381