(14) Specify the tilt series parameters including file name, tilt range, tilt increment. Also, take care to specify the image file format, and whether you wish
to save individual projections or an image stack (this is not critical, as it is
easy to convert between the two later).
(15) Record the tilt series and align and reconstruct using e.g. IMOD [142].
Usually, a tilt log of metadata will be saved along with the image stack,
specifying all acquisition conditions including tilt angles.
(16) Save aligned image stack (optional) and reconstructed volume.
(17) Optional steps: denoising*, segmentation, template matching*, subtomogram
averaging*
* applicable to cryo data only
References
1. S. Nickell et al., A visual approach to proteomics. Nat. Rev. Mol. Cell. Biol. 7(3), 225–230
(2006)
2. A. Hoenger, High-resolution cryo-electron microscopy on macromolecular complexes and
cell organelles. Protoplasma 251(2), 417–427 (2014)
3. Asano, S., B.D. Engel, and W. Baumeister, In Situ Cryo-Electron Tomography: A
Post-Reductionist Approach to Structural Biology. J. Mol. Biol. (2016). 428(2 Pt A): p. 332–
43
4. A. Leis et al., Visualizing cells at the nanoscale. Trends Biochem. Sci. 34(2), 60–70 (2009)
5. U.E. Maurer, B. Sodeik, K. Grunewald, Native 3D intermediates of membrane fusion in
herpes simplex virus 1 entry. Proc. Natl. Acad. Sci. U. S. A. 105(30), 10559–64 (2008)
6. K. Iwasaki, T. Omura, Electron tomography of the supramolecular structure of virus-infected
cells. Curr. Opin. Struct. Biol. 20(5), 632–9 (2010)
7. I. Ibiricu et al., Cryo electron tomography of herpes simplex virus during axonal transport
and secondary envelopment in primary neurons. PLoS Pathog. 7(12), e1002406 (2011)
8. C. Risco et al., Three-Dimensional Imaging of Viral Infections. Annu. Rev. Virol. 1(1), 453–
73 (2014)
9. S. Padilla-Parra, M. Tramier, FRET microscopy in the living cell: different approaches,
strengths and weaknesses. BioEssays. 34(5), 369–76 (2012)
10. K. Grunewald et al., Prospects of electron cryotomography to visualize macromolecular
complexes inside cellular compartments: implications of crowding. Biophys. Chem. 100(1–
3), 577–91 (2003)
11. G. Foffi et al., Macromolecular crowding: chemistry and physics meet biology (Ascona,
Switzerland, 10-14 June 2012). Phys. Biol. 10(4), 040301 (2013)
12. V. Lucic, A. Leis, W. Baumeister, Cryo-electron tomography of cells: connecting structure
and function. Histochem. Cell. Biol. 130(2), 185–96 (2008)
13. A.V. Agronskaia et al., Integrated fluorescence and transmission electron microscopy.
J. Struct. Biol. 164(2), 183–189 (2008)
14. K. Cortese, A. Diaspro, C. Tacchetti, Advanced correlative light/electron microscopy:
current methods and new developments using Tokuyasu cryosections. J. Histochem.
Cytochem. 57(12), 1103–12 (2009)
15. W. Kukulski et al., Correlated fluorescence and 3D electron microscopy with high sensitivity
and spatial precision. J. Cell. Biol. 192(1), 111–9 (2011)
16. R.I. Koning et al., Correlative cryo-fluorescence light microscopy and cryo-electron
tomography of Streptomyces. Methods Cell. Biol. 124, 217–39 (2014)
1 Electron Tomography: A Primer
27
to save individual projections or an image stack (this is not critical, as it is
easy to convert between the two later).
(15) Record the tilt series and align and reconstruct using e.g. IMOD [142].
Usually, a tilt log of metadata will be saved along with the image stack,
specifying all acquisition conditions including tilt angles.
(16) Save aligned image stack (optional) and reconstructed volume.
(17) Optional steps: denoising*, segmentation, template matching*, subtomogram
averaging*
* applicable to cryo data only
References
1. S. Nickell et al., A visual approach to proteomics. Nat. Rev. Mol. Cell. Biol. 7(3), 225–230
(2006)
2. A. Hoenger, High-resolution cryo-electron microscopy on macromolecular complexes and
cell organelles. Protoplasma 251(2), 417–427 (2014)
3. Asano, S., B.D. Engel, and W. Baumeister, In Situ Cryo-Electron Tomography: A
Post-Reductionist Approach to Structural Biology. J. Mol. Biol. (2016). 428(2 Pt A): p. 332–
43
4. A. Leis et al., Visualizing cells at the nanoscale. Trends Biochem. Sci. 34(2), 60–70 (2009)
5. U.E. Maurer, B. Sodeik, K. Grunewald, Native 3D intermediates of membrane fusion in
herpes simplex virus 1 entry. Proc. Natl. Acad. Sci. U. S. A. 105(30), 10559–64 (2008)
6. K. Iwasaki, T. Omura, Electron tomography of the supramolecular structure of virus-infected
cells. Curr. Opin. Struct. Biol. 20(5), 632–9 (2010)
7. I. Ibiricu et al., Cryo electron tomography of herpes simplex virus during axonal transport
and secondary envelopment in primary neurons. PLoS Pathog. 7(12), e1002406 (2011)
8. C. Risco et al., Three-Dimensional Imaging of Viral Infections. Annu. Rev. Virol. 1(1), 453–
73 (2014)
9. S. Padilla-Parra, M. Tramier, FRET microscopy in the living cell: different approaches,
strengths and weaknesses. BioEssays. 34(5), 369–76 (2012)
10. K. Grunewald et al., Prospects of electron cryotomography to visualize macromolecular
complexes inside cellular compartments: implications of crowding. Biophys. Chem. 100(1–
3), 577–91 (2003)
11. G. Foffi et al., Macromolecular crowding: chemistry and physics meet biology (Ascona,
Switzerland, 10-14 June 2012). Phys. Biol. 10(4), 040301 (2013)
12. V. Lucic, A. Leis, W. Baumeister, Cryo-electron tomography of cells: connecting structure
and function. Histochem. Cell. Biol. 130(2), 185–96 (2008)
13. A.V. Agronskaia et al., Integrated fluorescence and transmission electron microscopy.
J. Struct. Biol. 164(2), 183–189 (2008)
14. K. Cortese, A. Diaspro, C. Tacchetti, Advanced correlative light/electron microscopy:
current methods and new developments using Tokuyasu cryosections. J. Histochem.
Cytochem. 57(12), 1103–12 (2009)
15. W. Kukulski et al., Correlated fluorescence and 3D electron microscopy with high sensitivity
and spatial precision. J. Cell. Biol. 192(1), 111–9 (2011)
16. R.I. Koning et al., Correlative cryo-fluorescence light microscopy and cryo-electron
tomography of Streptomyces. Methods Cell. Biol. 124, 217–39 (2014)
1 Electron Tomography: A Primer
27