compared to bright-field TEM [19, 20], noting that phase plates now offer a solution
for the latter. A third TEM technique—single-particle analysis (SPA)—should be
considered separately as a powerful means of determining the structures of identical
or nearly identical copies of macromolecules or symmetrical (principally, icosahedral) viruses. SPA will be discussed briefly to introduce certain concepts, and for the
sake of completeness. The SEM tomography technique, serial block-face imaging,
utilises a scanning electron microscope in conjunction with a sectioning device,
either a diamond knife [21] or an ion beam [22–26], to build up a three-dimensional
volume, layer by layer, analogous to the operating principle of a confocal microscope. At present, this technique is practised on plastic-embedded specimens. It is
covered in detail in Chap. 5. To complete this short overview of tomography
strategies, it is worth mentioning the technique known as ‘array tomography’.
Although best known from fluorescence microscopy, it is equally applicable to the
serial-block-face technique or indeed, in combination (Chap. 6). It allows large-scale
surveys of classical ultrastructure afforded by serial block-face imaging but in
combination with the mapping of fluorescent epitopes. Like serial block-face
imaging, however, it is not suited to studies of molecular structural biology.
Finally, we can define a category of biophysical techniques that provide superior
resolution to electron tomography but with the caveat that the structures are generated from isolated and purified entities that are thus removed from the cellular
context. The resulting structures can be used for visual proteomics interrogations of
cellular interaction networks. These techniques are the standard techniques of
structural biology, and include X-ray crystallography as well as single-particle
analysis (cryo-) electron microscopy. As mentioned above, SPA is a reductionist
approach to solving structure that takes advantage of statistical sampling ex vivo.
The term ‘single-particle’ is a misnomer because the technique takes into account as
many particles as can be sampled efficiently, typically tens of thousands, rather than
a unique occurrence of a molecule within its native context. SPA is related to cryoelectron tomography in terms of common sample preparation and bright-field,
low-dose imaging conditions. It may begin with air-dried and stained preparations
because these structures serve as useful starting models for computational refinement of frozen-hydrated data. Unlike X-ray crystallography, SPA does not have a
requirement for crystals, and it can cope with large, multi-unit complexes as well as
some heterogeneity. For comprehensive reviews of SPA in structural biology and
virology, the reader is referred to detailed reviews [27–33]. Modern structural
biology increasingly aims to harness the synergies of these techniques when used in
combination (‘hybrid techniques’, [34]). In particular, the high-resolution structures
can be used to populate tomograms of cells via the process of pattern recognition in
real space (see ‘Template Matching’). Thus, it is important to consider tomograms
generated by electron tomography as complete maps of a cell’s proteome, where
(dose-) limited resolution can be circumvented to produce a ‘pseudo-atomic atlas’
of the macromolecular machinery that underlies cellular function [1].
The definition of electron tomography used in this book includes TEM/STEM
tomography as well as the newer SEM strategies. It therefore expands upon the
1 Electron Tomography: A Primer
3
for the latter. A third TEM technique—single-particle analysis (SPA)—should be
considered separately as a powerful means of determining the structures of identical
or nearly identical copies of macromolecules or symmetrical (principally, icosahedral) viruses. SPA will be discussed briefly to introduce certain concepts, and for the
sake of completeness. The SEM tomography technique, serial block-face imaging,
utilises a scanning electron microscope in conjunction with a sectioning device,
either a diamond knife [21] or an ion beam [22–26], to build up a three-dimensional
volume, layer by layer, analogous to the operating principle of a confocal microscope. At present, this technique is practised on plastic-embedded specimens. It is
covered in detail in Chap. 5. To complete this short overview of tomography
strategies, it is worth mentioning the technique known as ‘array tomography’.
Although best known from fluorescence microscopy, it is equally applicable to the
serial-block-face technique or indeed, in combination (Chap. 6). It allows large-scale
surveys of classical ultrastructure afforded by serial block-face imaging but in
combination with the mapping of fluorescent epitopes. Like serial block-face
imaging, however, it is not suited to studies of molecular structural biology.
Finally, we can define a category of biophysical techniques that provide superior
resolution to electron tomography but with the caveat that the structures are generated from isolated and purified entities that are thus removed from the cellular
context. The resulting structures can be used for visual proteomics interrogations of
cellular interaction networks. These techniques are the standard techniques of
structural biology, and include X-ray crystallography as well as single-particle
analysis (cryo-) electron microscopy. As mentioned above, SPA is a reductionist
approach to solving structure that takes advantage of statistical sampling ex vivo.
The term ‘single-particle’ is a misnomer because the technique takes into account as
many particles as can be sampled efficiently, typically tens of thousands, rather than
a unique occurrence of a molecule within its native context. SPA is related to cryoelectron tomography in terms of common sample preparation and bright-field,
low-dose imaging conditions. It may begin with air-dried and stained preparations
because these structures serve as useful starting models for computational refinement of frozen-hydrated data. Unlike X-ray crystallography, SPA does not have a
requirement for crystals, and it can cope with large, multi-unit complexes as well as
some heterogeneity. For comprehensive reviews of SPA in structural biology and
virology, the reader is referred to detailed reviews [27–33]. Modern structural
biology increasingly aims to harness the synergies of these techniques when used in
combination (‘hybrid techniques’, [34]). In particular, the high-resolution structures
can be used to populate tomograms of cells via the process of pattern recognition in
real space (see ‘Template Matching’). Thus, it is important to consider tomograms
generated by electron tomography as complete maps of a cell’s proteome, where
(dose-) limited resolution can be circumvented to produce a ‘pseudo-atomic atlas’
of the macromolecular machinery that underlies cellular function [1].
The definition of electron tomography used in this book includes TEM/STEM
tomography as well as the newer SEM strategies. It therefore expands upon the
1 Electron Tomography: A Primer
3