Chapter 4
Large-Scale Electron Tomography of Cells
Using SerialEM and IMOD
Eileen O’Toole, Peter van der Heide, J. Richard McIntosh
and David Mastronarde
Abstract We have developed methods to compute tomographic reconstructions of
relatively large areas and volumes of sectioned cells using SerialEM and IMOD.
The SerialEM program has emerged as a major tool for automated acquisition of tilt
series for electron tomography [1]. This program contains a number of unique
features, including a prediction algorithm that allows rapid image acquisition, and it
can also routinely acquire tilt series from montaged images. The IMOD package
contains alignment methods that correct for the non-uniform changes that occur
over a large specimen area during data collection [1, 2]. In addition, the IMOD
package offers tools for aligning and stacking tomograms from serial sections,
allowing larger volumes to be reconstructed. Until recently practical considerations,
such as constraints on electronic image shift, have limited the size of the area that
could be reconstructed using ordinary montaging to *10 Â 10 µm. The desire to
study areas larger than this has spurred the development of methods for stitching
together laterally adjacent tomograms, referred to as super-montaging. In this
chapter, we describe the steps involved in performing large-scale tomography
including super-montaging, using the human mitotic spindle as an example.
E. O’Toole (&) Á J. Richard McIntosh Á D. Mastronarde
Department of MCD Biology, University of Colorado, Boulder, CO, USA
e-mail: Eileen.otoole@colorado.edu
J. Richard McIntosh
e-mail: richard.mcintosh@colorado.edu
D. Mastronarde
e-mail: mast@Colorado.EDU
P. van der Heide
Institute for Molecular Bioscience, Queensland Bioscience Precinct,
The University of Queensland, Brisbane, Australia
© Springer International Publishing AG 2018
E. Hanssen (ed.), Cellular Imaging, Biological and Medical Physics,
Biomedical Engineering, https://doi.org/10.1007/978-3-319-68997-5_4
95
Large-Scale Electron Tomography of Cells
Using SerialEM and IMOD
Eileen O’Toole, Peter van der Heide, J. Richard McIntosh
and David Mastronarde
Abstract We have developed methods to compute tomographic reconstructions of
relatively large areas and volumes of sectioned cells using SerialEM and IMOD.
The SerialEM program has emerged as a major tool for automated acquisition of tilt
series for electron tomography [1]. This program contains a number of unique
features, including a prediction algorithm that allows rapid image acquisition, and it
can also routinely acquire tilt series from montaged images. The IMOD package
contains alignment methods that correct for the non-uniform changes that occur
over a large specimen area during data collection [1, 2]. In addition, the IMOD
package offers tools for aligning and stacking tomograms from serial sections,
allowing larger volumes to be reconstructed. Until recently practical considerations,
such as constraints on electronic image shift, have limited the size of the area that
could be reconstructed using ordinary montaging to *10 Â 10 µm. The desire to
study areas larger than this has spurred the development of methods for stitching
together laterally adjacent tomograms, referred to as super-montaging. In this
chapter, we describe the steps involved in performing large-scale tomography
including super-montaging, using the human mitotic spindle as an example.
E. O’Toole (&) Á J. Richard McIntosh Á D. Mastronarde
Department of MCD Biology, University of Colorado, Boulder, CO, USA
e-mail: Eileen.otoole@colorado.edu
J. Richard McIntosh
e-mail: richard.mcintosh@colorado.edu
D. Mastronarde
e-mail: mast@Colorado.EDU
P. van der Heide
Institute for Molecular Bioscience, Queensland Bioscience Precinct,
The University of Queensland, Brisbane, Australia
© Springer International Publishing AG 2018
E. Hanssen (ed.), Cellular Imaging, Biological and Medical Physics,
Biomedical Engineering, https://doi.org/10.1007/978-3-319-68997-5_4
95