[11, 13]. Although cryo-ET provides impressive detail of cellular
features, this method is not widely used by cell biologists mainly
due to the perceived complexity of the technique.
The basic workflow of a cryo-ET experiment involves the vitrification of samples, followed by data acquisition and processing
[14]. For the preservation of thin samples, e.g. purified proteins
or a monolayer of cells, vitrification can be achieved by plunging the
specimen surrounded by a thin layer of liquid into a cryogen
(e.g. liquid ethane). Excess liquid is blotted away using a filter
paper prior to plunging [8–12]. Devices for plunge freezing range
from custom-made manual plungers to commercial automated
instruments such as the Vitrobot (FEI, now Thermo Fisher), GP2
(Leica), or Cryo-Plunge 3 (Gatan). For thicker samples, e.g. small
multicellular organisms like C. elegans or tissue biopsies, highpressure freezing can be used to achieve vitrification
[15, 16]. The vitrified samples need to be kept below À140
C to
prevent devitrification, which damages samples through the formation of ice crystals. To prevent devitrification, samples are stored in
a liquid nitrogen dewar and are transferred under liquid nitrogen
conditions to the cryo-stage of a cryo-cooled transmission electron
microscope for imaging [14].
To perform cryo-ET, 2D projection images of the vitrified
sample are recorded at different tilt angles. This collection of
images is called a tilt series and normally covers a tilt range from
about À60
to +60
with an increment of typically 2
–3
per image
[17]. The single images are usually recorded at negative defocus
(~4–8 μm) [18, 19]. This allows images of sufficient contrast to be
generated but at the cost of resolution. If resolution is paramount, a
phase plate can be used which enhances contrast of close to focus
images [20, 21]. However, using a phase plate currently adds
complexity to data collection and processing.
Biological samples experience radiation damage when exposed
to an electron beam. Radiation damage causes the breakage of
chemical bonds and in severe cases, the complete destruction
(“boiling”) of a sample [22, 23]. To limit radiation damage, the
total electron dose that is applied to the sample needs to be limited
and controlled over the whole tilt series [14]. Tilt series are collected using a process called low dose where focusing is performed
off axis, and the area of interest is exposed at high magnifications
only when the image is being recorded, thus reducing the required
total electron dose [24, 25]. However, even under the best imaging
conditions, radiation damage will accumulate over the tilt series
range.
Sample imaging is also limited by poor electron penetration
depth, which directly affects obtainable resolution [13]. As a guide,
sample thickness should not exceed 600 nm for descent quality
tomograms when a 300 kV cryo-transmission electron microscope
(cryo-TEM) with energy filter is used. As the sample is tilted, its
4
Daniel Serwas and Karen M. Davies
features, this method is not widely used by cell biologists mainly
due to the perceived complexity of the technique.
The basic workflow of a cryo-ET experiment involves the vitrification of samples, followed by data acquisition and processing
[14]. For the preservation of thin samples, e.g. purified proteins
or a monolayer of cells, vitrification can be achieved by plunging the
specimen surrounded by a thin layer of liquid into a cryogen
(e.g. liquid ethane). Excess liquid is blotted away using a filter
paper prior to plunging [8–12]. Devices for plunge freezing range
from custom-made manual plungers to commercial automated
instruments such as the Vitrobot (FEI, now Thermo Fisher), GP2
(Leica), or Cryo-Plunge 3 (Gatan). For thicker samples, e.g. small
multicellular organisms like C. elegans or tissue biopsies, highpressure freezing can be used to achieve vitrification
[15, 16]. The vitrified samples need to be kept below À140
C to
prevent devitrification, which damages samples through the formation of ice crystals. To prevent devitrification, samples are stored in
a liquid nitrogen dewar and are transferred under liquid nitrogen
conditions to the cryo-stage of a cryo-cooled transmission electron
microscope for imaging [14].
To perform cryo-ET, 2D projection images of the vitrified
sample are recorded at different tilt angles. This collection of
images is called a tilt series and normally covers a tilt range from
about À60
to +60
with an increment of typically 2
–3
per image
[17]. The single images are usually recorded at negative defocus
(~4–8 μm) [18, 19]. This allows images of sufficient contrast to be
generated but at the cost of resolution. If resolution is paramount, a
phase plate can be used which enhances contrast of close to focus
images [20, 21]. However, using a phase plate currently adds
complexity to data collection and processing.
Biological samples experience radiation damage when exposed
to an electron beam. Radiation damage causes the breakage of
chemical bonds and in severe cases, the complete destruction
(“boiling”) of a sample [22, 23]. To limit radiation damage, the
total electron dose that is applied to the sample needs to be limited
and controlled over the whole tilt series [14]. Tilt series are collected using a process called low dose where focusing is performed
off axis, and the area of interest is exposed at high magnifications
only when the image is being recorded, thus reducing the required
total electron dose [24, 25]. However, even under the best imaging
conditions, radiation damage will accumulate over the tilt series
range.
Sample imaging is also limited by poor electron penetration
depth, which directly affects obtainable resolution [13]. As a guide,
sample thickness should not exceed 600 nm for descent quality
tomograms when a 300 kV cryo-transmission electron microscope
(cryo-TEM) with energy filter is used. As the sample is tilted, its
4
Daniel Serwas and Karen M. Davies