individual operations can also be provided to Etomo when starting to process a data
set interactively, in files referred to as templates. More than one template can be
used; for example, one file could specify parameters for a particular microscope,
and another could set parameters appropriate for cryoET.
The batch tomogram interface in Etomo has several components, on different
tabbed pages, for selecting the tilt series to process, setting parameters, and controlling and monitoring Batchruntomo as it runs. A detailed guide for its use can be
found at http://bio3d.colorado.edu/imod/betaDoc/batchGuide.html. Parameters and
operations are selected on a single page that presents a carefully chosen subset of
options. Less commonly needed options not exposed there can be entered through a
template file.
Processing through Batchruntomo is fully compatible with the interactive processing through Etomo. At the end of a batch run, the user can open any data set in
the reconstruction interface and examine the results, such as to see whether steps
such as fiducial tracking, alignment, or gold erasing were done correctly. The user
can also post-process the data (trim, scale or flatten the volume) and clean up
intermediate files. If a step is anticipated to need manual intervention in advance, it
is possible to run all sets through that step, check the results, and resume batch
processing. In general, very large reconstructions will require intervention more
often, but the batch interface is still useful for handling routine operations and
minimizing manual operations.
4.6 Using IMOD’s Join Interface to Stack Serial
Reconstructions
A limitation to ET is the section thickness one can image in the EM. The use of
higher voltage EMs (200–300 kV) allows significantly thicker sections to be
imaged (200–400 nm), depending on the density of material. However, at thicknesses greater than this, plural scattering and inelastic scattering events seriously
reduce image resolution. This is particularly a problem because the specimen
thickness doubles when the section is tilted to ±60°, and triples at 70° [14].
However, this limitation can be overcome by stacking, or joining tomograms
computed from serial sections, thus increasing the reconstructed sample volume.
The basic step in joining serial tomograms is to align a computed slice from the top
of one tomogram with a computed slice from the bottom of the adjacent tomogram.
Thanks to beam-induced distortions of each section, this alignment usually requires
more than a simple shift and rotation. A general linear transformation is typically
sufficient for smaller areas, but tomograms computed from larger, montaged areas
often contain non-linear differences between successive sections that must be
corrected with a warping transformation (see below). Once a transformation is
found for each section interface, these section-to-section transformations are converted into ones that bring all sections into alignment with each other with the
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E. O’Toole et al.
set interactively, in files referred to as templates. More than one template can be
used; for example, one file could specify parameters for a particular microscope,
and another could set parameters appropriate for cryoET.
The batch tomogram interface in Etomo has several components, on different
tabbed pages, for selecting the tilt series to process, setting parameters, and controlling and monitoring Batchruntomo as it runs. A detailed guide for its use can be
found at http://bio3d.colorado.edu/imod/betaDoc/batchGuide.html. Parameters and
operations are selected on a single page that presents a carefully chosen subset of
options. Less commonly needed options not exposed there can be entered through a
template file.
Processing through Batchruntomo is fully compatible with the interactive processing through Etomo. At the end of a batch run, the user can open any data set in
the reconstruction interface and examine the results, such as to see whether steps
such as fiducial tracking, alignment, or gold erasing were done correctly. The user
can also post-process the data (trim, scale or flatten the volume) and clean up
intermediate files. If a step is anticipated to need manual intervention in advance, it
is possible to run all sets through that step, check the results, and resume batch
processing. In general, very large reconstructions will require intervention more
often, but the batch interface is still useful for handling routine operations and
minimizing manual operations.
4.6 Using IMOD’s Join Interface to Stack Serial
Reconstructions
A limitation to ET is the section thickness one can image in the EM. The use of
higher voltage EMs (200–300 kV) allows significantly thicker sections to be
imaged (200–400 nm), depending on the density of material. However, at thicknesses greater than this, plural scattering and inelastic scattering events seriously
reduce image resolution. This is particularly a problem because the specimen
thickness doubles when the section is tilted to ±60°, and triples at 70° [14].
However, this limitation can be overcome by stacking, or joining tomograms
computed from serial sections, thus increasing the reconstructed sample volume.
The basic step in joining serial tomograms is to align a computed slice from the top
of one tomogram with a computed slice from the bottom of the adjacent tomogram.
Thanks to beam-induced distortions of each section, this alignment usually requires
more than a simple shift and rotation. A general linear transformation is typically
sufficient for smaller areas, but tomograms computed from larger, montaged areas
often contain non-linear differences between successive sections that must be
corrected with a warping transformation (see below). Once a transformation is
found for each section interface, these section-to-section transformations are converted into ones that bring all sections into alignment with each other with the
104
E. O’Toole et al.