4.8.2 Using IMOD’s Etomo Interface to Compute
Individual Montage Panels
Imod’s Etomo interface is used to compute tomograms from the individual montage tilt
series panels of the supermontage. Two practices can be important for facilitating the
lateral stitching. First, if the tilt axis is rotated more than *5° from vertical or horizontal, it is necessary to create oversized aligned stacks (where the tilt axis has been
rotated to be vertical) so that each tomogram includes all of the volume that can be
reconstructed with high quality. For the example in Fig. 4. 7b, a final size of 7000, 7000
pixels was used. If using the reconstruction interface, one can increase this size using
the advanced button in the final alignment page. To use automated processing with
Batchruntomo, the size would have to be set with a directive in a template file. The
second practice is to flatten the tomograms. Montaged tomograms often have a
dome-like or bent shape after reconstruction (Fig. 4.7c, top), possibly caused by the
gradients in lateral shrinkage between the area from which data was acquired, the larger
pre-irradiated area, and the surrounding unirradiated area. Tools for flattening volumes
are available in IMOD, where a model of the tomogram’s top and bottom surfaces is
generated and transformations are produced to flatten the material in the tomogram
(Fig. 4.7c, bottom). Flattening is useful for joining serial section tomograms, but we
have found it essential to do this step before lateral stitching of supermontage panels.
4.8.3 Using IMOD to Stitch Supermontages Laterally
The lateral edges of the resulting individual montages are stitched together to create
a super-montage using a series of programs that are run from a command line
(Fig. 4.8). The supermontage pipeline consists of five separate programs—
Setupstitch, Edgepatches, Fitpatches, Stitchalign, and Tomostitch—four of which
are Python scripts that run several other programs in IMOD [30]. Before beginning
the process, it is useful to make binned tomograms to test parameters and expedite
the process of creating the supermontage volume.
4.8.4 Setupstitch
The first tool in the pipeline is Setupstitch, which sets up the initial configuration file.
This file starts with information about the tomograms and their overlap; it is used and
added to by each program in the sequence. Details of the many options for this
program can be found at http://bio3d.colorado.edu/imod/doc/man/setupstitch.html.
Since oversized tomograms include regions that are empty of image contrast, and
areas reconstructed from incomplete data, a simple “patch region model” is created
to define the area containing useful data. An overlap between volumes at their edges
110
E. O’Toole et al.
Individual Montage Panels
Imod’s Etomo interface is used to compute tomograms from the individual montage tilt
series panels of the supermontage. Two practices can be important for facilitating the
lateral stitching. First, if the tilt axis is rotated more than *5° from vertical or horizontal, it is necessary to create oversized aligned stacks (where the tilt axis has been
rotated to be vertical) so that each tomogram includes all of the volume that can be
reconstructed with high quality. For the example in Fig. 4. 7b, a final size of 7000, 7000
pixels was used. If using the reconstruction interface, one can increase this size using
the advanced button in the final alignment page. To use automated processing with
Batchruntomo, the size would have to be set with a directive in a template file. The
second practice is to flatten the tomograms. Montaged tomograms often have a
dome-like or bent shape after reconstruction (Fig. 4.7c, top), possibly caused by the
gradients in lateral shrinkage between the area from which data was acquired, the larger
pre-irradiated area, and the surrounding unirradiated area. Tools for flattening volumes
are available in IMOD, where a model of the tomogram’s top and bottom surfaces is
generated and transformations are produced to flatten the material in the tomogram
(Fig. 4.7c, bottom). Flattening is useful for joining serial section tomograms, but we
have found it essential to do this step before lateral stitching of supermontage panels.
4.8.3 Using IMOD to Stitch Supermontages Laterally
The lateral edges of the resulting individual montages are stitched together to create
a super-montage using a series of programs that are run from a command line
(Fig. 4.8). The supermontage pipeline consists of five separate programs—
Setupstitch, Edgepatches, Fitpatches, Stitchalign, and Tomostitch—four of which
are Python scripts that run several other programs in IMOD [30]. Before beginning
the process, it is useful to make binned tomograms to test parameters and expedite
the process of creating the supermontage volume.
4.8.4 Setupstitch
The first tool in the pipeline is Setupstitch, which sets up the initial configuration file.
This file starts with information about the tomograms and their overlap; it is used and
added to by each program in the sequence. Details of the many options for this
program can be found at http://bio3d.colorado.edu/imod/doc/man/setupstitch.html.
Since oversized tomograms include regions that are empty of image contrast, and
areas reconstructed from incomplete data, a simple “patch region model” is created
to define the area containing useful data. An overlap between volumes at their edges
110
E. O’Toole et al.