definition provided in the most recent textbook on electron tomography [18], which
suggests that it should loosely incorporate strategies where the specimen is tilted.
The revised definition reflects the recent success of approaches for building up 3D
volumes by SEM, which was previously regarded almost exclusively as a ‘surface’
technique. This chapter will introduce the principles of TEM- and SEM-based
electron tomography, recent technological developments, and selected contributions
to cell and structural biology. It cannot possibly do justice to the published
applications of electron tomography. For recent, general reviews, the reader is
referred to references [3, 35]. Other recent reviews cover application of electron
tomography to viruses [30, 36] and prokaryotes [37], while newer publications
enabled by low-temperature nanofabrication showcase novel insights and possibilities for the biology of ‘higher’ organisms [38–41].
1.2 Principles of Electron Tomography
The TEM strategy for electron tomography is analogous to medical X-ray imaging
(computed tomography—‘CT’ or ‘CAT’ scans) in that it uses projections of a
specimen recorded from different viewing angles and back-projects them into
Fourier Space (syn. frequency domain) to calculate a real-space (syn. position
space) reconstruction of the ‘specimen’ (Fig. 1.2). The main difference to computed
tomography in terms of image acquisition is the tilting of the specimen in electron
tomography compared to tilting of the imaging gantry during computed tomography, although a concept for a tilting electron microscope has been published [42].
Note also that ‘soft’ X-ray tomography of cells utilises a fixed X-ray source or
synchrotron beamline and either a rotating or tilting sample holder. A TEM
goniometer and sample holder are used for the latter. TEM tomography is used
routinely for specimens that have undergone a substitution process to replace the
sample’s water with a plastic resin, which in turn can be sectioned and introduced
into the microscope using a standard sample holder. This technique is useful for
studies of ultrastructure, such as the arrangements of organelles [43] or studies of
e.g. membranes during virus morphogenesis [44, 45]. A variation of this protocol
with additional contrast agent is used for serial-block-face SEM tomography [46]
(see Chap. 5 and below). Cryo- electron tomography (or electron cryotomography,
Chap. 3) is the method of choice for appropriately thin, frozen-hydrated specimens,
where structural accuracy is paramount, i.e. the density map reflects the true nature
of the feature rather than added contrast agent, the accumulation of which depends
on its relative affinity for the different regions of a molecule.
So-called ‘single-particle analysis’ 3D electron microscopy uses projections of
many copies of identical or nearly identical, frozen-hydrated macromolecules or
viruses to build up a 3D reconstruction. This powerful, biochemical reductionist
approach combines computational averaging of many copies of identical or nearly
identical, randomly orientated molecules with statistical suppression of noise to
generate a composite structure of the molecule at a resolution typically better than
4
A. Leis
suggests that it should loosely incorporate strategies where the specimen is tilted.
The revised definition reflects the recent success of approaches for building up 3D
volumes by SEM, which was previously regarded almost exclusively as a ‘surface’
technique. This chapter will introduce the principles of TEM- and SEM-based
electron tomography, recent technological developments, and selected contributions
to cell and structural biology. It cannot possibly do justice to the published
applications of electron tomography. For recent, general reviews, the reader is
referred to references [3, 35]. Other recent reviews cover application of electron
tomography to viruses [30, 36] and prokaryotes [37], while newer publications
enabled by low-temperature nanofabrication showcase novel insights and possibilities for the biology of ‘higher’ organisms [38–41].
1.2 Principles of Electron Tomography
The TEM strategy for electron tomography is analogous to medical X-ray imaging
(computed tomography—‘CT’ or ‘CAT’ scans) in that it uses projections of a
specimen recorded from different viewing angles and back-projects them into
Fourier Space (syn. frequency domain) to calculate a real-space (syn. position
space) reconstruction of the ‘specimen’ (Fig. 1.2). The main difference to computed
tomography in terms of image acquisition is the tilting of the specimen in electron
tomography compared to tilting of the imaging gantry during computed tomography, although a concept for a tilting electron microscope has been published [42].
Note also that ‘soft’ X-ray tomography of cells utilises a fixed X-ray source or
synchrotron beamline and either a rotating or tilting sample holder. A TEM
goniometer and sample holder are used for the latter. TEM tomography is used
routinely for specimens that have undergone a substitution process to replace the
sample’s water with a plastic resin, which in turn can be sectioned and introduced
into the microscope using a standard sample holder. This technique is useful for
studies of ultrastructure, such as the arrangements of organelles [43] or studies of
e.g. membranes during virus morphogenesis [44, 45]. A variation of this protocol
with additional contrast agent is used for serial-block-face SEM tomography [46]
(see Chap. 5 and below). Cryo- electron tomography (or electron cryotomography,
Chap. 3) is the method of choice for appropriately thin, frozen-hydrated specimens,
where structural accuracy is paramount, i.e. the density map reflects the true nature
of the feature rather than added contrast agent, the accumulation of which depends
on its relative affinity for the different regions of a molecule.
So-called ‘single-particle analysis’ 3D electron microscopy uses projections of
many copies of identical or nearly identical, frozen-hydrated macromolecules or
viruses to build up a 3D reconstruction. This powerful, biochemical reductionist
approach combines computational averaging of many copies of identical or nearly
identical, randomly orientated molecules with statistical suppression of noise to
generate a composite structure of the molecule at a resolution typically better than
4
A. Leis