higher zoom of the boxed areas in Fig. 4.5a, misalignments are evident in other
areas of the images. Warping points were added (Fig. 4.5d, red stars) and warping
alignments were applied at these local regions, greatly improving the overall
alignment across the entire area (Fig. 4.5e, f). The alignment transforms are then
applied to the serial tomograms creating a final, joined volume.
4.7 Using IMOD’s 3dmod for Modeling Features in Serial
Reconstructions
Tomographic volumes can be viewed and structures of interest modeled using IMOD’s
3dmod program. A detailed guide on its use can be found at http://bio3d.colorado.edu/
imod/doc/3dmodguide.html. 3dmod contains useful features for modeling membranes,
vesicles, and trajectories such as microtubules and filaments. One tool, the slicer
window, allows the user to rotate the position of a slice cut for display from the image
data to get the best view of a particular object. This slicer window is particularly useful
for tracking complex networks of microtubules in the mitotic spindle (Fig. 4.6).
Tomograms were computed from 3 Â 3 montage tilt series (Fig. 4.6a) from five,
serial, 300 nm sections. Individual microtubules were tracked throughout the volume
(Fig. 4.6b). The complete models shown in Fig. 4.6c, d are based on a reconstructed
volume of 7 Â 7 Â 1.5 µm
3
. A total of 1402 microtubules were modeled in this
volume. The left panel shows kinetochore-associated microtubules (KMTs, yellow),
non-KMTs (red or white, depending on their likely pole association), and centrioles
(blue). Light blue and green dots mark pole-distal and pole-proximal microtubule ends.
The right panel shows only the microtubules that were situated with both ends in the
reconstructed volume. The differences between the left and right panels demonstrate
the value of working towards complete spindle reconstructions: when many microtubules leave the reconstructed volume, the information about microtubule lengths and
end locations is compromised.
Quantitative measurements of spindle characteristics, such as microtubule
lengths and 3D microtubule end positions, can then be measured from model
contour data, using programs in the IMOD software package. An initial quantitative
analysis of the spindle microtubules in these reconstructions shows the differences
in KMT and non-KMT lengths and end distributions (Fig. 4.6e). Analysis of these
differences is sure to be informative about the dynamics of different microtubule
classes.
3dmod has image caching capabilities that allow one to work with volumes that
are too large to fit into memory. The most advanced caching feature provides rapid
access to very large volumes at a full range of zooms by working with an “image
pyramid”, which consists of copies of the volume at different resolutions. Such a
pyramid can be built with a simple IMOD command.
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E. O’Toole et al.
areas of the images. Warping points were added (Fig. 4.5d, red stars) and warping
alignments were applied at these local regions, greatly improving the overall
alignment across the entire area (Fig. 4.5e, f). The alignment transforms are then
applied to the serial tomograms creating a final, joined volume.
4.7 Using IMOD’s 3dmod for Modeling Features in Serial
Reconstructions
Tomographic volumes can be viewed and structures of interest modeled using IMOD’s
3dmod program. A detailed guide on its use can be found at http://bio3d.colorado.edu/
imod/doc/3dmodguide.html. 3dmod contains useful features for modeling membranes,
vesicles, and trajectories such as microtubules and filaments. One tool, the slicer
window, allows the user to rotate the position of a slice cut for display from the image
data to get the best view of a particular object. This slicer window is particularly useful
for tracking complex networks of microtubules in the mitotic spindle (Fig. 4.6).
Tomograms were computed from 3 Â 3 montage tilt series (Fig. 4.6a) from five,
serial, 300 nm sections. Individual microtubules were tracked throughout the volume
(Fig. 4.6b). The complete models shown in Fig. 4.6c, d are based on a reconstructed
volume of 7 Â 7 Â 1.5 µm
3
. A total of 1402 microtubules were modeled in this
volume. The left panel shows kinetochore-associated microtubules (KMTs, yellow),
non-KMTs (red or white, depending on their likely pole association), and centrioles
(blue). Light blue and green dots mark pole-distal and pole-proximal microtubule ends.
The right panel shows only the microtubules that were situated with both ends in the
reconstructed volume. The differences between the left and right panels demonstrate
the value of working towards complete spindle reconstructions: when many microtubules leave the reconstructed volume, the information about microtubule lengths and
end locations is compromised.
Quantitative measurements of spindle characteristics, such as microtubule
lengths and 3D microtubule end positions, can then be measured from model
contour data, using programs in the IMOD software package. An initial quantitative
analysis of the spindle microtubules in these reconstructions shows the differences
in KMT and non-KMT lengths and end distributions (Fig. 4.6e). Analysis of these
differences is sure to be informative about the dynamics of different microtubule
classes.
3dmod has image caching capabilities that allow one to work with volumes that
are too large to fit into memory. The most advanced caching feature provides rapid
access to very large volumes at a full range of zooms by working with an “image
pyramid”, which consists of copies of the volume at different resolutions. Such a
pyramid can be built with a simple IMOD command.
106
E. O’Toole et al.