Skip to main content
SpringerLink
Log in
Book cover

Cellular Imaging pp 95–116Cite as

Large-Scale Electron Tomography of Cells Using SerialEM and IMOD

  • Chapter
  • First Online:

Part of the book series: Biological and Medical Physics, Biomedical Engineering ((BIOMEDICAL))

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 × 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.

This is a preview of subscription content, log in via an institution.

References

  1. D.N. Mastronarde, Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152(1), 36–51 (2005)

    Article  Google Scholar 

  2. J.R. Kremer, D.N. Mastronarde, J.R. McIntosh, Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116(1), 71–76 (1995)

    Article  Google Scholar 

  3. C.J. Peddie, L.M. Collinson, Exploring the third dimension: volume electron microscopy comes of age. Micron 61, 9–19 (2014)

    Article  Google Scholar 

  4. A. Kremer, S. Lippens, S. Bartunkova, B. Asselbergh, C. Blanpain, M. Fendrych, A. Goossens, M. Holt, S. Janssens, M. Krols, J.-C. Larsimont, C. Mc Guire, M.K. Nowack, X. Saelens, A. Schertel, B. Schepens, M. Slezak, V. Timmerman, C. Theunis, R. VAN Brempt, Y. Visser, C.J. Guérin, Developing 3D SEM in a broad biological context. J. Microsc. 259(2), 80–96 (2015)

    Article  Google Scholar 

  5. J.R. McIntosh, K.L. McDonald, M.K. Edwards, B.M. Ross, Three-dimensional structure of the central mitotic spindle of Diatoma vulgare. J. Cell Biol. 83(2 Pt 1), 428–442 (1979)

    Article  Google Scholar 

  6. J.G. White, E. Southgate, J.N. Thomson, S. Brenner, The structure of the nervous system of the nematode Caenorhabditis elegans. Philos. Trans. R. Soc. Lond. B Biol. Sci. 314(1165), 1–340 (1986)

    Article  ADS  Google Scholar 

  7. S. Takemura, Connectome of the fly visual circuitry. Reprod. Syst. Sex. Disord. 64(1), 37–44 (2015)

    Google Scholar 

  8. J.R. Anderson, B.W. Jones, C.B. Watt, M.V. Shaw, J.-H. Yang, D. Demill, J.S. Lauritzen, Y. Lin, K.D. Rapp, D. Mastronarde, P. Koshevoy, B. Grimm, T. Tasdizen, R. Whitaker, R.E. Marc, Exploring the retinal connectome. Mol. Vis. 17, 355–379 (2011)

    Google Scholar 

  9. K.J. Hayworth, J.L. Morgan, R. Schalek, D.R. Berger, D.G.C. Hildebrand, J.W. Lichtman, Imaging ATUM ultrathin section libraries with WaferMapper: a multi-scale approach to EM reconstruction of neural circuits. Front. Neural Circuits 8, 68 (2014)

    Article  Google Scholar 

  10. K.D. Micheva, S.J. Smith, Array tomography: a new tool for imaging the molecular architecture and ultrastructure of neural circuits. Neuron 55(1), 25–36 (2007)

    Article  Google Scholar 

  11. N. Kasthuri, K.J. Hayworth, D.R. Berger, R.L. Schalek, J.A. Conchello, S. Knowles-Barley, D. Lee, A. Vázquez-Reina, V. Kaynig, T.R. Jones, M. Roberts, J.L. Morgan, J.C. Tapia, H.S. Seung, W.G. Roncal, J.T. Vogelstein, R. Burns, D.L. Sussman, C.E. Priebe, H. Pfister, J.W. Lichtman, Saturated reconstruction of a volume of neocortex. Cell 162(3), 648–661 (2015)

    Article  Google Scholar 

  12. W. Denk, H. Horstmann, Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol. 2(11), e329 (2004)

    Article  Google Scholar 

  13. D. Wei, S. Jacobs, S. Modla, S. Zhang, C.L. Young, R. Cirino, J. Caplan, K. Czymmek, High-resolution three-dimensional reconstruction of a whole yeast cell using focused-ion beam scanning electron microscopy. Biotechniques 53(1), 41–48 (2012)

    Google Scholar 

  14. R. McIntosh, D. Nicastro, D. Mastronarde, New views of cells in 3D: an introduction to electron tomography. Trends Cell Biol. 15(1), 43–51 (2005)

    Article  Google Scholar 

  15. A. Hoenger, J.R. McIntosh, Probing the macromolecular organization of cells by electron tomography. Curr. Opin. Cell Biol. 21(1), 89–96 (2009)

    Article  Google Scholar 

  16. V. Lučič, A. Rigort, W. Baumeister, Cryo-electron tomography: the challenge of doing structural biology in situ. J. Cell Biol. 202(3), 407–419 (2013)

    Article  Google Scholar 

  17. G.E. Soto, S.J. Young, M.E. Martone, T.J. Deerinck, S. Lamont, B.O. Carragher, K. Hama, M.H. Ellisman, Serial section electron tomography: a method for three-dimensional reconstruction of large structures. Neuroimage 1(3), 230–243 (1994)

    Article  Google Scholar 

  18. M.S. Ladinsky, D.N. Mastronarde, J.R. McIntosh, K.E. Howell, L.A. Staehelin, Golgi structure in three dimensions: functional insights from the normal rat kidney cell. J. Cell Biol. 144(6), 1135–1149 (1999)

    Article  Google Scholar 

  19. A.B. Noske, A.J. Costin, G.P. Morgan, B.J. Marsh, Expedited approaches to whole cell electron tomography and organelle mark-up in situ in high-pressure frozen pancreatic islets. J. Struct. Biol. 161(3), 298–313 (2008)

    Article  Google Scholar 

  20. J.L. Höög, C. Schwartz, A.T. Noon, E.T. O’Toole, D.N. Mastronarde, J.R. McIntosh, C. Antony, Organization of interphase microtubules in fission yeast analyzed by electron tomography. Dev. Cell 12(3), 349–361 (2007)

    Article  Google Scholar 

  21. S. Redemann, T. Müller-Reichert, Correlative light and electron microscopy for the analysis of cell division. J. Microsc. 251(2), 109–112 (2013)

    Article  Google Scholar 

  22. S. Phan, A. Lawrence, T. Molina, J. Lanman, M. Berlanga, M. Terada, A. Kulungowski, J. Obayashi, M. Ellisman, TxBR montage reconstruction for large field electron tomography. J. Struct. Biol. 180, 154–164 (2012)

    Article  Google Scholar 

  23. D.N. Mastronarde, Dual-axis tomography: an approach with alignment methods that preserve resolution. J. Struct. Biol. 120(3), 343–352 (1997)

    Article  Google Scholar 

  24. K.L. McDonald, M. Morphew, P. Verkade, T. Müller-Reichert, Recent advances in high-pressure freezing: equipment- and specimen-loading methods. Methods Mol. Biol. 369, 143–173 (2007)

    Article  Google Scholar 

  25. K. McDonald, H. Schwarz, T. Müller-Reichert, R. Webb, C. Buser, M. Morphew, ‘Tips and tricks’ for high-pressure freezing of model systems. Methods Cell Biol. 96, 671–693 (2010)

    Article  Google Scholar 

  26. Q.S. Zheng, M.B. Braunfeld, J.W. Sedat, D.A. Agard, An improved strategy for automated electron microscopic tomography. J. Struct. Biol. 147(2), 91–101 (2004)

    Article  Google Scholar 

  27. S. Nickell, F. Förster, A. Linaroudis, W. Del Net, F. Beck, R. Hegerl, W. Baumeister, J.M. Plitzko, TOM software toolbox: acquisition and analysis for electron tomography. J. Struct. Biol. 149(3), 227–234 (2005)

    Article  Google Scholar 

  28. D. Mastronarde, Automated tomographic reconstruction in the IMOD software package. Microsc. Micronal. 19(Suppl. 2), 544–545 (2013)

    Article  ADS  MathSciNet  Google Scholar 

  29. D.N. Mastronarde, S.R. Held, Automated tilt series alignment and tomographic reconstruction in IMOD. J. Struct. Biol. 197(2), 102–113 (2017)

    Google Scholar 

  30. D.N. Mastronarde, P.A. van der Heide, G.P. Morgan, B.J. Marsh, Supermontaging: Reconstructing large cellular volumes by stitching together laterally adjacent tomograms. Microsc. Microanal. 14(Suppl2), 106–107 (2008)

    Article  Google Scholar 

  31. M.K. Morphew, 3D immunolocalization with plastic sections. Methods Cell Biol. 79, 493–513 (2007)

    Article  Google Scholar 

  32. T. Müller-Reichert, I. Sassoon, E. O’Toole, M. Romao, A.J. Ashford, A.A. Hyman, C. Antony, Analysis of the distribution of the kinetochore protein Ndc10p in Saccharomyces cerevisiae using 3-D modeling of mitotic spindles. Chromosoma 111(7), 417–428 (2003)

    Article  Google Scholar 

  33. A.J. Bridge, M. Morphew, R. Bartlett, I.M. Hagan, The fission yeast SPB component Cut12 links bipolar spindle formation to mitotic control. Genes Dev. 12(7), 927–942 (1998)

    Article  Google Scholar 

  34. B.-H. Kang, E. Nielsen, M.L. Preuss, D. Mastronarde, L.A. Staehelin, Electron tomography of RabA4b- and PI-4 Kβ1-labeled trans Golgi network compartments in Arabidopsis. Traffic 12(3), 313–329 (2011)

    Article  Google Scholar 

  35. X. Shu, V. Lev-Ram, T.J. Deerinck, Y. Qi, E.B. Ramko, M.W. Davidson, Y. Jin, M.H. Ellisman, R.Y. Tsien, A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol. 9(4), e1001041 (2011)

    Article  Google Scholar 

  36. J.D. Martell, T.J. Deerinck, Y. Sancak, T.L. Poulos, V.K. Mootha, G.E. Sosinsky, M.H. Ellisman, A.Y. Ting, Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat. Biotechnol. 30(11), 1143–1148 (2012)

    Article  Google Scholar 

  37. W. Kukulski, M. Schorb, M. Kaksonen, J.A.G. Briggs, Plasma membrane reshaping during endocytosis is revealed by time-resolved electron tomography. Cell 150(3), 508–520 (2012)

    Article  Google Scholar 

  38. W. Kukulski, M. Schorb, S. Welsch, A. Picco, M. Kaksonen, J.A.G. Briggs, Precise, correlated fluorescence microscopy and electron tomography of lowicryl sections using fluorescent fiducial markers. Methods Cell Biol. 111, 235–257 (2012)

    Article  Google Scholar 

  39. M.K. Morphew, E.T. O’Toole, C.L. Page, M. Pagratis, J. Meehl, T. Giddings, J.M. Gardner, C. Ackerson, S.L. Jaspersen, M. Winey, A. Hoenger, J.R. McIntosh, Metallothionein as a clonable tag for protein localization by electron microscopy of cells. J. Microsc. 260(1), 20–29 (2015)

    Article  Google Scholar 

Download references

Acknowledgements

The authors would like to dedicate this chapter to the life and memory of Peter van der Heide who was instrumental in the early development of these methods. We would like to thank Mary Morphew for specimen preparation. This work was supported by and NIH RR00592 and P41GM103431 to A. Hoenger and by NIH/NIBIB grant R01-EB005027 to D. N. Mastronarde

Author information

Authors and Affiliations

  1. Department of MCD Biology, University of Colorado, Boulder, CO, USA

    Eileen O’Toole, J. Richard McIntosh & David Mastronarde

  2. Institute for Molecular Bioscience, Queensland Bioscience Precinct, The University of Queensland, Brisbane, Australia

    Peter van der Heide

Authors
  1. Eileen O’Toole
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Peter van der Heide
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J. Richard McIntosh
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David Mastronarde
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Eileen O’Toole .

Editor information

Editors and Affiliations

  1. Bio21 Institute Advanced Microscopy Facility, University of Melbourne, Melbourne, Victoria, Australia

    Eric Hanssen

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer International Publishing AG

About this chapter

Check for updates. Verify currency and authenticity via CrossMark

Cite this chapter

O’Toole, E., van der Heide, P., Richard McIntosh, J., Mastronarde, D. (2018). Large-Scale Electron Tomography of Cells Using SerialEM and IMOD. In: Hanssen, E. (eds) Cellular Imaging. Biological and Medical Physics, Biomedical Engineering. Springer, Cham. https://doi.org/10.1007/978-3-319-68997-5_4

Download citation

Publish with us

Policies and ethics

Search

Navigation