increased reproducibility; blotting parameters typically tuned are blot time and
position of blotting paper to control wicking of excess liquid from the grid, and
chamber temperature and humidity to regulate evaporation between blotting
and plunge-freezing (evaporation rapidly increases the osmotic pressure in a thin
film, so can dehydrate cells and disrupt proteins). After vitrification, grids can be
immediately transferred to the microscope, placed in long-term storage under liquid
nitrogen, or shipped in a dry-shipper to another facility.
Vitrification remains as much an art as a science, however, with ice quality
depending on subtleties of the specimen, liquid medium, grid manufacturer and
hydrophilicity of the support. Reliably reproducing good grids remains a bottleneck
for many projects, and future developments will enable routine and reproducible
grid preparation. As such, it is important to screen vitrification conditions for each
new specimen type. Screening is best performed on a screening microscope with
the ability to rapidly load single grids via a side-entry cryoholder; the speed of such
a system enables the user to shuttle between the microscope and vitrification
apparatus, iteratively refining vitrification parameters.
3.3.2 Vitrifying Thick Specimens: Strategies Towards
Tractability
Although specimens as thick as a few microns can be vitrified without formation of
crystalline ice, specimens thicker than *500 nm suffer from excessive inelastic and
multiple scattering that progressively degrade the image signal-to-noise ratio,
requiring thinning approaches even in high-powered microscopes. There are currently two techniques to thin a specimen after freezing: the relatively well-developed
cryo-sectioning of specimens using a diamond blade cooled to cryo temperatures
(CEMOVIS), and milling techniques to slice through the specimen using a focused
ion beam.
Specimens too thick for vitrification by plunge-freezing can fortunately be vitrified under high-pressures (2100 bar) to lower the freezing point of water and slow
the formation of ice [28]. A cryo-microtome can subsequently be used to cut the
pellet into thin slices (typically 50–200 nm thick) to be manipulated onto a grid for
ECT [29]. Cryo-sectioning is technically challenging and can be hindered by artefacts such as compression crevasses caused by the action of the sectioning blade.
An approach suitable for specimens thin enough for freezing at ambient temperatures on a grid, yet too thick for imaging whole, is to use a focused ion beam
(FIB) to mill away biological material above and below the part of the specimen of
interest. An ion beam—typically of Gallium ions [30]—is directed to mill a plane
through the specimen nearly parallel to the grid plane, leaving a thin lamellum
supported by unmilled specimen edges, allowing for tilt series acquisition.
FIB-milling is a multi-step process, involving ablating the bulk of the specimen first
with a high current, followed by a low-current polishing step, minimizing radiation
damage to the surface of the lamella [31]. The approach is not currently
high-throughput, although is undergoing rapid development [32].
3 Electron Cryo-Tomography
67
position of blotting paper to control wicking of excess liquid from the grid, and
chamber temperature and humidity to regulate evaporation between blotting
and plunge-freezing (evaporation rapidly increases the osmotic pressure in a thin
film, so can dehydrate cells and disrupt proteins). After vitrification, grids can be
immediately transferred to the microscope, placed in long-term storage under liquid
nitrogen, or shipped in a dry-shipper to another facility.
Vitrification remains as much an art as a science, however, with ice quality
depending on subtleties of the specimen, liquid medium, grid manufacturer and
hydrophilicity of the support. Reliably reproducing good grids remains a bottleneck
for many projects, and future developments will enable routine and reproducible
grid preparation. As such, it is important to screen vitrification conditions for each
new specimen type. Screening is best performed on a screening microscope with
the ability to rapidly load single grids via a side-entry cryoholder; the speed of such
a system enables the user to shuttle between the microscope and vitrification
apparatus, iteratively refining vitrification parameters.
3.3.2 Vitrifying Thick Specimens: Strategies Towards
Tractability
Although specimens as thick as a few microns can be vitrified without formation of
crystalline ice, specimens thicker than *500 nm suffer from excessive inelastic and
multiple scattering that progressively degrade the image signal-to-noise ratio,
requiring thinning approaches even in high-powered microscopes. There are currently two techniques to thin a specimen after freezing: the relatively well-developed
cryo-sectioning of specimens using a diamond blade cooled to cryo temperatures
(CEMOVIS), and milling techniques to slice through the specimen using a focused
ion beam.
Specimens too thick for vitrification by plunge-freezing can fortunately be vitrified under high-pressures (2100 bar) to lower the freezing point of water and slow
the formation of ice [28]. A cryo-microtome can subsequently be used to cut the
pellet into thin slices (typically 50–200 nm thick) to be manipulated onto a grid for
ECT [29]. Cryo-sectioning is technically challenging and can be hindered by artefacts such as compression crevasses caused by the action of the sectioning blade.
An approach suitable for specimens thin enough for freezing at ambient temperatures on a grid, yet too thick for imaging whole, is to use a focused ion beam
(FIB) to mill away biological material above and below the part of the specimen of
interest. An ion beam—typically of Gallium ions [30]—is directed to mill a plane
through the specimen nearly parallel to the grid plane, leaving a thin lamellum
supported by unmilled specimen edges, allowing for tilt series acquisition.
FIB-milling is a multi-step process, involving ablating the bulk of the specimen first
with a high current, followed by a low-current polishing step, minimizing radiation
damage to the surface of the lamella [31]. The approach is not currently
high-throughput, although is undergoing rapid development [32].
3 Electron Cryo-Tomography
67