amenable to large-volume imaging. It is beyond the scope of this introductory
chapter to outline the theory of scanning electron microscopy and focussed ion
beam technology, suffice to say that the sample preparation closely resembles that
of plastic blocks used for TEM tomography but with the further incorporation of/
doping with heavy metals to aid in contrast and to obviate specimen charging [24,
25, 46]. The appearance of the resulting micrographs also resembles bright-field
TEM micrographs. This familiar appearance means that interpretation and segmentation tasks are similar to TEM tomography of plastic sections [84] but the
strategy is applicable to a much larger scale, such as whole cells. The approach is
described in detail in Chap. 5
It is also worth noting at this point that scanning electron microscopes using an
in-built ultramicrotome, the so-called ‘Denk technique’ [21], require a variable
pressure or ‘environmental’ SEM (VP-SEM, ESEM, respectively). This is to cope
with outgassing from each newly exposed resin surface, which has been discussed
above in relation to mass loss. Ablation with a focussed ion beam requires a
dedicated ‘dual-beam’ or ‘cross-beam’ FIB-SEM instrument. This is a scanning EM
instrument with an ion beam column fitted at a fixed angle to the electron gun (the
stage, however, can typically be tilted and possibly also rotated to provide maximum freedom for nanofabrication). Finally, the FIB-SEM with its highly flexible
milling capabilities can be used to nanofabricate specimens for cryo- electron
tomography [39, 40, 66, 67], provided that the vitrification cold-chain is uninterrupted, and that the specimen is protected from the formation of frost during transfer
to the TEM. Electron diffraction studies by Marko and colleagues [85] show
convincingly that the milling process does not compromise vitrification. Areas
adjacent to FIB-milled lamellae are sputtered destructively, which highlights the
importance of preserving target structures such that they are located within the
remaining volume.
1.7 Alignment of Projections
A TEM projection micrograph from a thin specimen contains a wealth of 3D
information, yet in the classical 2D depiction, features from multiple planes are
superimposed, and the ability to determine relative depth and thereby distinguish
between interconnected and discrete features is lost. For example, features may
appear to be contiguous when they are in fact separate, unconnected entities lying in
close proximity. The true nature of the sample densities and their spatial relationships become apparent when an aligned tilt series of projections is viewed as an
image sequence. The current discussion refers almost exclusively to alignment of
projections acquired by TEM tomography, where alignment is a mandatory step of
the workflow. (N.B. The term ‘alignment’ is essentially synonymous with the term
‘registration’ used commonly in medical imaging.)
Unlike some related imaging techniques that operate in Fourier space, e.g.
magnetic resonance imaging (MRI), individual projections from a TEM tilt series
1 Electron Tomography: A Primer
13
chapter to outline the theory of scanning electron microscopy and focussed ion
beam technology, suffice to say that the sample preparation closely resembles that
of plastic blocks used for TEM tomography but with the further incorporation of/
doping with heavy metals to aid in contrast and to obviate specimen charging [24,
25, 46]. The appearance of the resulting micrographs also resembles bright-field
TEM micrographs. This familiar appearance means that interpretation and segmentation tasks are similar to TEM tomography of plastic sections [84] but the
strategy is applicable to a much larger scale, such as whole cells. The approach is
described in detail in Chap. 5
It is also worth noting at this point that scanning electron microscopes using an
in-built ultramicrotome, the so-called ‘Denk technique’ [21], require a variable
pressure or ‘environmental’ SEM (VP-SEM, ESEM, respectively). This is to cope
with outgassing from each newly exposed resin surface, which has been discussed
above in relation to mass loss. Ablation with a focussed ion beam requires a
dedicated ‘dual-beam’ or ‘cross-beam’ FIB-SEM instrument. This is a scanning EM
instrument with an ion beam column fitted at a fixed angle to the electron gun (the
stage, however, can typically be tilted and possibly also rotated to provide maximum freedom for nanofabrication). Finally, the FIB-SEM with its highly flexible
milling capabilities can be used to nanofabricate specimens for cryo- electron
tomography [39, 40, 66, 67], provided that the vitrification cold-chain is uninterrupted, and that the specimen is protected from the formation of frost during transfer
to the TEM. Electron diffraction studies by Marko and colleagues [85] show
convincingly that the milling process does not compromise vitrification. Areas
adjacent to FIB-milled lamellae are sputtered destructively, which highlights the
importance of preserving target structures such that they are located within the
remaining volume.
1.7 Alignment of Projections
A TEM projection micrograph from a thin specimen contains a wealth of 3D
information, yet in the classical 2D depiction, features from multiple planes are
superimposed, and the ability to determine relative depth and thereby distinguish
between interconnected and discrete features is lost. For example, features may
appear to be contiguous when they are in fact separate, unconnected entities lying in
close proximity. The true nature of the sample densities and their spatial relationships become apparent when an aligned tilt series of projections is viewed as an
image sequence. The current discussion refers almost exclusively to alignment of
projections acquired by TEM tomography, where alignment is a mandatory step of
the workflow. (N.B. The term ‘alignment’ is essentially synonymous with the term
‘registration’ used commonly in medical imaging.)
Unlike some related imaging techniques that operate in Fourier space, e.g.
magnetic resonance imaging (MRI), individual projections from a TEM tilt series
1 Electron Tomography: A Primer
13