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Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells

Nature Protocols volume 12, pages 150–167 (2017)Cite this article

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Abstract

Correlative light and electron microscopy (CLEM) combines spatiotemporal information from fluorescence light microscopy (fLM) with high-resolution structural data from cryo-electron tomography (cryo-ET). These technologies provide opportunities to bridge knowledge gaps between cell and structural biology. Here we describe our protocol for correlated cryo-fLM, cryo-electron microscopy (cryo-EM), and cryo-ET (i.e., cryo-CLEM) of virus-infected or transfected mammalian cells. Mammalian-derived cells are cultured on EM substrates, using optimized conditions that ensure that the cells are spread thinly across the substrate and are not physically disrupted. The cells are then screened by fLM and vitrified before acquisition of cryo-fLM and cryo-ET images, which is followed by data processing. A complete session from grid preparation through data collection and processing takes 5–15 d for an individual experienced in cryo-EM.

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Figure 1: Flowchart of the steps for CLEM of mammalian cells.
Figure 2: Use of fLM to determine cross-sectional cell thickness and cell permissivity to RSV.
Figure 3: Carbon evaporation onto gold Finder EM grids.
Figure 4: Representative light microscopy image of the ideal cell density present on an EM grid.
Figure 5: Gatan Cryoplunge3 system setup for plunge-freezing.
Figure 6: Leica EM Cryo CLEM system.
Figure 7: Cryo-fluorescence microscopy grid map of HIV-1 virus-like particles tethered to HT1080 cells collected using the Leica LASX software.
Figure 8: Cryo-TEM map of grid with coordinates imposed from Leica LASX software.
Figure 9: Entire cryo-CLEM imaging workflow with an HIV-1 Gag-tetherin specimen.
Figure 10: Cryo-CLEM imaging of retroviral endocytosis and fusion.
Figure 11: Montage maps provide cellular context for cryo-ET data.
Figure 12: Quantitative segmentation data of HIV-1 particles with tetherin.
Figure 13: Examples of poor-quality grids for cryo-CLEM imaging.

References

  1. Morgan, C., Godman, G.C., Breitenfeld, P.M. & Rose, H.M. A correlative study by electron and light microscopy of the development of type 5 adenovirus. I. Electron microscopy. J. Exp. Med. 112, 373–382 (1960).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Godman, G.C., Morgan, C., Breitenfeld, P.M. & Rose, H.M. A correlative study by electron and light microscopy of the development of type 5 adenovirus. II. Light microscopy. J. Exp. Med. 112, 383–402 (1960).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Dubochet, J. et al. Cryo-electron microscopy of vitrified specimens. Q. Rev. Biophys. 21, 129–228 (1988).

    Article  CAS  PubMed  Google Scholar 

  4. Mancini, E.J., de Haas, F. & Fuller, S.D. High-resolution icosahedral reconstruction: fulfilling the promise of cryo-electron microscopy. Structure 5, 741–750 (1997).

    Article  CAS  PubMed  Google Scholar 

  5. Steven, A.C. & Aebi, U. The next ice age: cryo-electron tomography of intact cells. Trends Cell Biol. 13, 107–110 (2003).

    Article  CAS  PubMed  Google Scholar 

  6. Risco, C. & Carrascosa, J.L. Visualization of viral assembly in the infected cell. Histol. Histopathol. 14, 905–926 (1999).

    CAS  PubMed  Google Scholar 

  7. Schroder, R.R. Advances in electron microscopy: a qualitative view of instrumentation development for macromolecular imaging and tomography. Arch. Biochem. Biophys. 581, 25–38 (2015).

    Article  PubMed  CAS  Google Scholar 

  8. McDonald, K.L. A review of high-pressure freezing preparation techniques for correlative light and electron microscopy of the same cells and tissues. J. Microsc. 235, 273–281 (2009).

    Article  CAS  PubMed  Google Scholar 

  9. Carroni, M. & Saibil, H.R. Cryo electron microscopy to determine the structure of macromolecular complexes. Methods 95, 78–85 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Giepmans, B.N., Adams, S.R., Ellisman, M.H. & Tsien, R.Y. The fluorescent toolbox for assessing protein location and function. Science 312, 217–224 (2006).

    Article  CAS  PubMed  Google Scholar 

  11. Desai, T.M. et al. Fluorescent protein-tagged Vpr dissociates from HIV-1 core after viral fusion and rapidly enters the cell nucleus. Retrovirology 12, 88 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Padilla-Parra, S. et al. Fusion of mature HIV-1 particles leads to complete release of a gag-GFP-based content marker and raises the intraviral pH. PLoS One 8, e71002 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Briegel, A. et al. Correlated light and electron cryo-microscopy. Methods Enzymol. 481, 317–341 (2010).

    Article  PubMed  Google Scholar 

  14. Strauss, J.D. et al. Three-dimensional structural characterization of HIV-1 tethered to human cells. J. Virol. 90, 1507–1521 (2015).

    Article  PubMed  CAS  Google Scholar 

  15. Yi, H. et al. Native immunogold labeling of cell surface proteins and viral glycoproteins for cryo-electron microscopy and cryo-electron tomography applications. J. Histochem. Cytochem. 63, 780–792 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  Google Scholar 

  17. Bharat, T.A., Russo, C.J., Lowe, J., Passmore, L.A. & Scheres, S.H. Advances in single-particle electron cryomicroscopy structure determination applied to sub-tomogram averaging. Structure 23, 1743–1753 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Hrabe, T. Localize.pytom: a modern webserver for cryo-electron tomography. Nucleic Acids Res. 43, W231–236 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Huiskonen, J.T. et al. Averaging of viral envelope glycoprotein spikes from electron cryotomography reconstructions using Jsubtomo. J. Vis. Exp. e51714 (2014).

  20. Heymann, J.B., Cardone, G., Winkler, D.C. & Steven, A.C. Computational resources for cryo-electron tomography in Bsoft. J. Struct. Biol. 161, 232–242 (2008).

    Article  PubMed  Google Scholar 

  21. Castano-Diez, D., Kudryashev, M., Arheit, M. & Stahlberg, H. Dynamo: a flexible, user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments. J. Struct. Biol. 178, 139–151 (2012).

    Article  PubMed  Google Scholar 

  22. Koning, R.I. et al. Correlative cryo-fluorescence light microscopy and cryo-electron tomography of Streptomyces. Methods Cell Biol. 124, 217–239 (2014).

    Article  PubMed  Google Scholar 

  23. van Driel, L.F., Valentijn, J.A., Valentijn, K.M., Koning, R.I. & Koster, A.J. Tools for correlative cryo-fluorescence microscopy and cryo-electron tomography applied to whole mitochondria in human endothelial cells. Eur. J. Cell Biol. 88, 669–684 (2009).

    Article  CAS  PubMed  Google Scholar 

  24. Jun, S. et al. Direct visualization of HIV-1 with correlative live-cell microscopy and cryo-electron tomography. Structure 19, 1573–1581 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Ibiricu, I. et al. Cryo electron tomography of herpes simplex virus during axonal transport and secondary envelopment in primary neurons. PLoS Pathog. 7, e1002406 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Schorb, M. & Briggs, J.A. Correlated cryo-fluorescence and cryo-electron microscopy with high spatial precision and improved sensitivity. Ultramicroscopy 143, 24–32 (2014).

    Article  CAS  PubMed  Google Scholar 

  27. Briegel, A. et al. Location and architecture of the Caulobacter crescentus chemoreceptor array. Mol. Microbiol. 69, 30–41 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Swulius, M.T. et al. Long helical filaments are not seen encircling cells in electron cryotomograms of rod-shaped bacteria. Biochem. Biophys. Res. Commun. 407, 650–655 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Chang, Y.W. et al. Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography. Nat. Methods 11, 737–739 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Wang, Q., Mercogliano, C.P. & Lowe, J. A ferritin-based label for cellular electron cryotomography. Structure 19, 147–154 (2011).

    Article  CAS  PubMed  Google Scholar 

  31. Gold, V.A. et al. Visualizing active membrane protein complexes by electron cryotomography. Nat. Commun. 5, 4129 (2014).

    Article  CAS  PubMed  Google Scholar 

  32. Mercogliano, C.P. & DeRosier, D.J. Concatenated metallothionein as a clonable gold label for electron microscopy. J. Struct. Biol. 160, 70–82 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Al-Amoudi, A. et al. Cryo-electron microscopy of vitreous sections. EMBO J. 23, 3583–3588 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Gruska, M., Medalia, O., Baumeister, W. & Leis, A. Electron tomography of vitreous sections from cultured mammalian cells. J. Struct. Biol. 161, 384–392 (2008).

    Article  CAS  PubMed  Google Scholar 

  35. Engel, B.D. et al. Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography. Elife 4, e04889 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Marko, M., Hsieh, C., Schalek, R., Frank, J. & Mannella, C. Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy. Nat. Methods 4, 215–217 (2007).

    Article  CAS  PubMed  Google Scholar 

  37. Wolf, S.G., Houben, L. & Elbaum, M. Cryo-scanning transmission electron tomography of vitrified cells. Nat. Methods 11, 423–428 (2014).

    Article  CAS  PubMed  Google Scholar 

  38. Bykov, Y.S., Cortese, M., Briggs, J.A. & Bartenschlager, R. Correlative light and electron microscopy methods for the study of virus-cell interactions. FEBS Lett. 590, 1877–1895 (2016).

    Article  CAS  PubMed  Google Scholar 

  39. Schellenberger, P. et al. High-precision correlative fluorescence and electron cryo microscopy using two independent alignment markers. Ultramicroscopy 143, 41–51 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Liu, B. et al. Three-dimensional super-resolution protein localization correlated with vitrified cellular context. Sci. Rep. 5, 13017 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Wolff, G., Hagen, C., Grunewald, K. & Kaufmann, R. Towards correlative super-resolution fluorescence and electron cryo-microscopy. Biol. Cell 108, 245–258 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Schwartz, C.L., Sarbash, V.I., Ataullakhanov, F.I., McIntosh, J.R. & Nicastro, D. Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching. J. Microsc. 227, 98–109 (2007).

    Article  PubMed  Google Scholar 

  43. Sartori, A. et al. Correlative microscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography. J. Struct. Biol. 160, 135–145 (2007).

    Article  PubMed  Google Scholar 

  44. Rigort, A. et al. Micromachining tools and correlative approaches for cellular cryo-electron tomography. J. Struct. Biol. 172, 169–179 (2010).

    Article  PubMed  Google Scholar 

  45. Faas, F.G. et al. Localization of fluorescently labeled structures in frozen-hydrated samples using integrated light electron microscopy. J. Struct. Biol. 181, 283–290 (2013).

    Article  CAS  PubMed  Google Scholar 

  46. Schorb, M. et al. New hardware and workflows for semi-automated correlative cryo-fluorescence and cryo-electron microscopy/tomography. J. Struct. Biol. https://dx.doi.org/10.1016/j.jsb.2016.06.020 (2016).

  47. Galaz-Montoya, J.G., Flanagan, J., Schmid, M.F. & Ludtke, S.J. Single particle tomography in EMAN2. J. Struct. Biol. 190, 279–290 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007).

    Article  CAS  PubMed  Google Scholar 

  49. Nicastro, D. et al. The molecular architecture of axonemes revealed by cryoelectron tomography. Science 313, 944–948 (2006).

    Article  CAS  PubMed  Google Scholar 

  50. Heumann, J.M., Hoenger, A. & Mastronarde, D.N. Clustering and variance maps for cryo-electron tomography using wedge-masked differences. J. Struct. Biol. 175, 288–299 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  51. Winkler, H. 3D reconstruction and processing of volumetric data in cryo-electron tomography. J. Struct. Biol. 157, 126–137 (2007).

    Article  CAS  PubMed  Google Scholar 

  52. Huiskonen, J.T. et al. Electron cryotomography of Tula hantavirus suggests a unique assembly paradigm for enveloped viruses. J. Virol. 84, 4889–4897 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Kremer, J.R., Mastronarde, D.N. & McIntosh, J.R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).

    Article  CAS  PubMed  Google Scholar 

  54. Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  55. Hagen, W.J., Wan, W. & Briggs, J.A. Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging. J. Struct. Biol. http://dx.doi/org/10.1016/j.jsb.2016.06.007 (2016).

  56. Moerner, W.E. & Orrit, M. Illuminating single molecules in condensed matter. Science 283, 1670–1676 (1999).

    Article  CAS  PubMed  Google Scholar 

  57. Kaufmann, R., Hagen, C. & Grunewald, K. Fluorescence cryo-microscopy: current challenges and prospects. Curr. Opin. Chem. Biol. 20, 86–91 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Xiong, Q., Morphew, M.K., Schwartz, C.L., Hoenger, A.H. & Mastronarde, D.N. CTF determination and correction for low dose tomographic tilt series. J. Struct. Biol. 168, 378–387 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  59. Heymann, J.B. & Belnap, D.M. Bsoft: image processing and molecular modeling for electron microscopy. J. Struct. Biol. 157, 3–18 (2007).

    Article  CAS  PubMed  Google Scholar 

  60. Heymann, J.B. Bsoft: image and molecular processing in electron microscopy. J. Struct. Biol. 133, 156–169 (2001).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank the Robert P. Apkarian Integrated Electron Microscopy Core of Emory University for microscopy services and support. This work was supported in part by grants from Emory University, Children's Healthcare of Atlanta, and the Georgia Research Alliance to E.R.W.; a grant from the Center for AIDS Research at Emory University (P30 AI050409); a grant from the James B. Pendleton Charitable Trust to E.R.W. and P.W.S.; public health service grants R01GM104540, R21AI101775, and R01GM104540-03S1 from the NIH to E.R.W.; NSF grant 0923395 to E.R.W.; public health service grant R01GM114561 from the NIH to E.R.W. and P.J.S.; public health service grant R01AI058828 from the NIH to P.W.S.; public health service grants R01GM054787 and R01AI053668 from the NIH to G.B.M.; public health service grant R01GM094198 from the NIH to P.J.S.; and public health service grant F32GM112517 from the NIH to J.D.S. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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Author notes

  1. Cheri M Hampton, Joshua D Strauss, Zunlong Ke and Rebecca S Dillard: These authors contributed equally to this work.

  2. erwrigh@emory.edu

Authors and Affiliations

  1. Division of Infectious Diseases, Department of Pediatrics, Emory University School of Medicine, Children's Healthcare of Atlanta, Atlanta, Georgia, USA

    Cheri M Hampton, Joshua D Strauss, Rebecca S Dillard, Jason E Hammonds, Tanay M Desai, Mariana Marin, Rachel E Storms, Fredrick Leon, Gregory B Melikyan, Paul W Spearman & Elizabeth R Wright

  2. School of Biology, Georgia Institute of Technology, Atlanta, Georgia, USA

    Zunlong Ke

  3. Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA

    Eric Alonas & Philip J Santangelo

  4. Robert P. Apkarian Integrated Electron Microscopy Core, Emory University, Atlanta, Georgia, USA

    Elizabeth R Wright

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Contributions

C.M.H. and E.R.W. wrote the manuscript. C.M.H., J.D.S., Z.K., R.S.D., J.E.H., E.A., T.M.D., M.M., G.B.M., P.J.S., P.W.S., and E.R.W. designed and performed the experiments, and edited the manuscript. R.E.S. and F.L. processed and analyzed data. All authors read and approved the manuscript.

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Hampton, C., Strauss, J., Ke, Z. et al. Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells. Nat Protoc 12, 150–167 (2017). https://doi.org/10.1038/nprot.2016.168

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