14. Press ‘Anchor Map’. The program automatically makes that image a map and
acquires the medium-magnification map.
15. Repeat steps 12–14 until you have found all of your areas of interest.
16. Select the first tilt series map and check ‘Tilt series’ in the Navigator window.
– this will prompt to name the file and set up the tilt series parameters.
19. Make each subsequent tilt series area a TS.
– it is possible to change individual tilt series parameters if they differ from
the original. The file names will automatically be chosen.
20. In Navigator drop down menu, select “Acquire at Points”.
21. Select ‘Autocenter beam’ to invoke a beam-centering procedure; ‘Cook specimen’ if the area has not been pre-irradiated, and ‘Fine eucentricity’ if this
operation is not selected in the tilt series parameters for each series. Set ‘Close
valves at end’ and ‘Send email’ as desired, then press the GO button. Both
‘Autocenter beam’ and ‘Cook specimen’ require setting up conditions for the
operation in advance.
4.4 Using IMOD’s Etomo Interface to Compute Basic
Single Frame or Montage Tilt Series
The IMOD package is a suite of programs used to compute tomographic reconstructions and to model cellular features. It is freely available on our website (http://
bio3d.colorado.edu/imod) and there are tutorials and guides for its use. IMOD runs
on several platforms (Linux, Mac OS X, Windows) making this a flexible and
powerful option for tomographic studies. The basic flow of operation is shown in the
flow chart in Fig. 4.4 and is similar to the process control buttons in the Etomo
graphical user interface that is used to create tomograms. The basic steps are:
(1) preprocess the tilt series data to remove the extreme pixel intensities generated by
x-rays, (2) create an approximately aligned stack and a fiducial model for alignment,
(3) refine the alignment, (4) set angular and position parameters to reconstruct the
specimen in the smallest volume, (5) create a final aligned stack and generate the
tomogram, (6) combine tomograms from tilt series taken around two orthogonal
axes; and, (7) trim the final tomogram and warp it if necessary so that the specimen is
flat. We encourage users first to familiarize themselves with the steps involved in
building dual axis tomograms through use of a tutorial before building their own
tomograms: http://bio3d.colorado.edu/imod/doc/etomoTutorial.html.
Virtually all of these steps can run automatically, and the Etomo interface allows
the user to take advantage of this automation for many of the steps (Fig. 4.4). For
example, the fiducial model can be generated automatically [28], and tomograms
can be positioned and combined automatically, which can greatly reduce the effort
required to go through the process.
102
E. O’Toole et al.
acquires the medium-magnification map.
15. Repeat steps 12–14 until you have found all of your areas of interest.
16. Select the first tilt series map and check ‘Tilt series’ in the Navigator window.
– this will prompt to name the file and set up the tilt series parameters.
19. Make each subsequent tilt series area a TS.
– it is possible to change individual tilt series parameters if they differ from
the original. The file names will automatically be chosen.
20. In Navigator drop down menu, select “Acquire at Points”.
21. Select ‘Autocenter beam’ to invoke a beam-centering procedure; ‘Cook specimen’ if the area has not been pre-irradiated, and ‘Fine eucentricity’ if this
operation is not selected in the tilt series parameters for each series. Set ‘Close
valves at end’ and ‘Send email’ as desired, then press the GO button. Both
‘Autocenter beam’ and ‘Cook specimen’ require setting up conditions for the
operation in advance.
4.4 Using IMOD’s Etomo Interface to Compute Basic
Single Frame or Montage Tilt Series
The IMOD package is a suite of programs used to compute tomographic reconstructions and to model cellular features. It is freely available on our website (http://
bio3d.colorado.edu/imod) and there are tutorials and guides for its use. IMOD runs
on several platforms (Linux, Mac OS X, Windows) making this a flexible and
powerful option for tomographic studies. The basic flow of operation is shown in the
flow chart in Fig. 4.4 and is similar to the process control buttons in the Etomo
graphical user interface that is used to create tomograms. The basic steps are:
(1) preprocess the tilt series data to remove the extreme pixel intensities generated by
x-rays, (2) create an approximately aligned stack and a fiducial model for alignment,
(3) refine the alignment, (4) set angular and position parameters to reconstruct the
specimen in the smallest volume, (5) create a final aligned stack and generate the
tomogram, (6) combine tomograms from tilt series taken around two orthogonal
axes; and, (7) trim the final tomogram and warp it if necessary so that the specimen is
flat. We encourage users first to familiarize themselves with the steps involved in
building dual axis tomograms through use of a tutorial before building their own
tomograms: http://bio3d.colorado.edu/imod/doc/etomoTutorial.html.
Virtually all of these steps can run automatically, and the Etomo interface allows
the user to take advantage of this automation for many of the steps (Fig. 4.4). For
example, the fiducial model can be generated automatically [28], and tomograms
can be positioned and combined automatically, which can greatly reduce the effort
required to go through the process.
102
E. O’Toole et al.