of the beam in “Exposure” mode and the square at its center
represents the detector, the green circle represents the size of the
beam in “Tracking” mode and the blue circle represents the size of
the beam in “Focus” mode. Select the green circle and move it
along the tilt-axis (the only direction it will move) to a region that
does not overlap the region of interest or another target area. Move
the blue circle to overlap with the green one. Ensure that the ice is
not too thick in this region. These are the regions where the
tracking and autofocus images will be collected, so it is important
that there is good contrast for the features to track easily. In the
“batch” section of the interface, give the file a name and set the
defocus (see Note 13). Press the “Add Position” button to add this
location to the list of tilt-series. Move to the next saved target and
repeat the process until all the desired targets have been added.
Once you have all of the tilt-series positions added, go to the “View
Positions” section of the “Tomography” tab. Press the “Refine All”
button on the menu bar. This procedure will have the scope revisit
each position, perform an auto eucentric height and focus, and
collect reference images for several imaging modes that it will use
during data collection to find and center the target again. It takes
~5 min per target position. Positions that cannot be refined can be
deleted from the list by right clicking on them and selecting
“Delete”. Before deleting, it can be useful to right click and on
the position in the list and “refine” again. Observe where the
procedure fails and decide if there is something that can be done
to fix the issue.
3.6.8 Start Data
Collection
In the “Tilt-Series” section of the “Tomography” tab, press the
“Parameters” button and fill out the fields. Table 2 (left side)
contains an example of values appropriate for data collection of
neuronal branches in culture. Once the parameters are specified,
press the “Corrections” button and fill out the fields. Table 2 (right
side) contains the values appropriate for data collection of neuronal
branches in culture. Press the “Acquire” button. The dataset will
collect automatically, but it is recommended to observe the first tiltseries begin with no errors.
3.7 Preprocessing
and Reconstruction
This section covers the basics of how to reconstruct tomograms
using the “Etomo” program within the IMOD image processing
suite [9] (https://bio3d.colorado.edu/imod/). While there are
multiple packages for doing tomographic reconstruction, IMOD
has long been many labs’ program of choice due to the fact that:
(a) It is well maintained and easy to install/update, (b) the documentation is extensive and the developers are fast to respond, and
(c) it is capable of reconstructing tomograms individually and in
batches. In addition to this outline, it is recommended that the
official IMOD guide be consulted when necessary at http://bio3d.
colorado.edu/imod/doc/tomoguide.html. Unless otherwise specified, the default values are used.
36
Ryan K. Hylton et al.
represents the detector, the green circle represents the size of the
beam in “Tracking” mode and the blue circle represents the size of
the beam in “Focus” mode. Select the green circle and move it
along the tilt-axis (the only direction it will move) to a region that
does not overlap the region of interest or another target area. Move
the blue circle to overlap with the green one. Ensure that the ice is
not too thick in this region. These are the regions where the
tracking and autofocus images will be collected, so it is important
that there is good contrast for the features to track easily. In the
“batch” section of the interface, give the file a name and set the
defocus (see Note 13). Press the “Add Position” button to add this
location to the list of tilt-series. Move to the next saved target and
repeat the process until all the desired targets have been added.
Once you have all of the tilt-series positions added, go to the “View
Positions” section of the “Tomography” tab. Press the “Refine All”
button on the menu bar. This procedure will have the scope revisit
each position, perform an auto eucentric height and focus, and
collect reference images for several imaging modes that it will use
during data collection to find and center the target again. It takes
~5 min per target position. Positions that cannot be refined can be
deleted from the list by right clicking on them and selecting
“Delete”. Before deleting, it can be useful to right click and on
the position in the list and “refine” again. Observe where the
procedure fails and decide if there is something that can be done
to fix the issue.
3.6.8 Start Data
Collection
In the “Tilt-Series” section of the “Tomography” tab, press the
“Parameters” button and fill out the fields. Table 2 (left side)
contains an example of values appropriate for data collection of
neuronal branches in culture. Once the parameters are specified,
press the “Corrections” button and fill out the fields. Table 2 (right
side) contains the values appropriate for data collection of neuronal
branches in culture. Press the “Acquire” button. The dataset will
collect automatically, but it is recommended to observe the first tiltseries begin with no errors.
3.7 Preprocessing
and Reconstruction
This section covers the basics of how to reconstruct tomograms
using the “Etomo” program within the IMOD image processing
suite [9] (https://bio3d.colorado.edu/imod/). While there are
multiple packages for doing tomographic reconstruction, IMOD
has long been many labs’ program of choice due to the fact that:
(a) It is well maintained and easy to install/update, (b) the documentation is extensive and the developers are fast to respond, and
(c) it is capable of reconstructing tomograms individually and in
batches. In addition to this outline, it is recommended that the
official IMOD guide be consulted when necessary at http://bio3d.
colorado.edu/imod/doc/tomoguide.html. Unless otherwise specified, the default values are used.
36
Ryan K. Hylton et al.