Joining of super-montages from adjacent serial sections is then performed using
non-linear alignments of the adjacent serial sections. Such non-linear alignments are
essential for aligning super-montage tomograms because the effects of non-uniform
changes in the specimen during data collection are amplified over such large areas.
This is done using warping transforms, as illustrated in Fig. 4.5, along with the help
of segmented objects that span from one section to the next, providing registration
points for the alignment of the successive reconstructions. In this example, half of
an anaphase spindle was reconstructed from 3 serial sections with a total size of
14.75 lm  6.14 lm  900 nm. A complete 1 lm mitotic spindle from budding
yeast is shown for comparison (Fig. 4.7d; inset). Individual microtubules can be
modeled and tracked in the volume and different classes of microtubules can be
defined, such as those that connect to the chromosomes (Fig. 4.7, light blue). Even
in these large volumes, details of the morphology of open and closed MT ends in
the spindle can be resolved (Fig. 4.7d, right).
4.9 Conclusions and Future Directions
Large-scale EM tomography makes it possible to study the 3D organization of
complex cellular assemblies, such as the mitotic spindle, with an almost isotropic
resolution that is not attainable by other methods. Automation of image acquisition
using SerialEM and options for complete automation of tomographic reconstruction
using IMOD greatly facilitate the various operations and reduce the time it takes to
collect the necessary data and compute the 3D volumes. A major advantage of
large-scale tomography over other volume EM methods is its ability to image fine
structure details at *4 nm resolution in their cellular context following optimal
fixation procedures, such as rapid freezing/freeze-substitution, and without excessive staining. The time intensive nature of large-scale tomography and its expense
are limitations to the method, but it is currently the best approach for visualizing
fine structural details, such as membrane coats, microtubule end morphologies, and
very slender filaments over larger cellular volumes.
A major goal for future 3-D EM studies is the development of electron-dense
labels that can be used for the localization of specific proteins in well-preserved
samples. In the past, studies coupling ssTEM with immunocytochemistry have been
used to document the spatial distribution of cellular antigens [31]. This approach
has been used to study the localization of specific proteins throughout the yeast
mitotic spindle as well as the yeast centrosome [32, 33]. Similarly, immunoelectron
tomography has been used to identify antigens directly below the section surface to
confirm the identity of proteins associated with the Golgi membranes in plant cells
[34]. However, these methods have limitations in that only those antigens present at
the surface of the section are labeled, reducing the z-resolution. Several groups have
employed small, clonable polypeptide to tag proteins with a label that can use either
catalysis [35] or photo-activation [36] to oxidize diaminobenzidine and produce a
polymer that becomes electron dense upon fixation with OsO 4 . These methods have
4 Large-Scale Electron Tomography of Cells …
113
non-linear alignments of the adjacent serial sections. Such non-linear alignments are
essential for aligning super-montage tomograms because the effects of non-uniform
changes in the specimen during data collection are amplified over such large areas.
This is done using warping transforms, as illustrated in Fig. 4.5, along with the help
of segmented objects that span from one section to the next, providing registration
points for the alignment of the successive reconstructions. In this example, half of
an anaphase spindle was reconstructed from 3 serial sections with a total size of
14.75 lm  6.14 lm  900 nm. A complete 1 lm mitotic spindle from budding
yeast is shown for comparison (Fig. 4.7d; inset). Individual microtubules can be
modeled and tracked in the volume and different classes of microtubules can be
defined, such as those that connect to the chromosomes (Fig. 4.7, light blue). Even
in these large volumes, details of the morphology of open and closed MT ends in
the spindle can be resolved (Fig. 4.7d, right).
4.9 Conclusions and Future Directions
Large-scale EM tomography makes it possible to study the 3D organization of
complex cellular assemblies, such as the mitotic spindle, with an almost isotropic
resolution that is not attainable by other methods. Automation of image acquisition
using SerialEM and options for complete automation of tomographic reconstruction
using IMOD greatly facilitate the various operations and reduce the time it takes to
collect the necessary data and compute the 3D volumes. A major advantage of
large-scale tomography over other volume EM methods is its ability to image fine
structure details at *4 nm resolution in their cellular context following optimal
fixation procedures, such as rapid freezing/freeze-substitution, and without excessive staining. The time intensive nature of large-scale tomography and its expense
are limitations to the method, but it is currently the best approach for visualizing
fine structural details, such as membrane coats, microtubule end morphologies, and
very slender filaments over larger cellular volumes.
A major goal for future 3-D EM studies is the development of electron-dense
labels that can be used for the localization of specific proteins in well-preserved
samples. In the past, studies coupling ssTEM with immunocytochemistry have been
used to document the spatial distribution of cellular antigens [31]. This approach
has been used to study the localization of specific proteins throughout the yeast
mitotic spindle as well as the yeast centrosome [32, 33]. Similarly, immunoelectron
tomography has been used to identify antigens directly below the section surface to
confirm the identity of proteins associated with the Golgi membranes in plant cells
[34]. However, these methods have limitations in that only those antigens present at
the surface of the section are labeled, reducing the z-resolution. Several groups have
employed small, clonable polypeptide to tag proteins with a label that can use either
catalysis [35] or photo-activation [36] to oxidize diaminobenzidine and produce a
polymer that becomes electron dense upon fixation with OsO 4 . These methods have
4 Large-Scale Electron Tomography of Cells …
113