Article | Published:

High-speed nanoscopic tracking of the position and orientation of a single virus

Nature Methods volume 6, pages 923927 (2009) | Download Citation

Abstract

Optical studies have revealed that, after binding, virions move laterally on the plasma membrane, but the complexity of the cellular environment and the drawbacks of fluorescence microscopy have prevented access to the molecular dynamics of early virus-host couplings, which are important for cell infection. Here we present a colocalization methodology that combines scattering interferometry and single-molecule fluorescence microscopy to visualize both position and orientation of single quantum dot–labeled Simian virus 40 (SV40) particles. By achieving nanometer spatial and 8 ms temporal resolution, we observed sliding and tumbling motions during rapid lateral diffusion on supported lipid bilayers, and repeated back and forth rocking between nanoscopic regions separated by 9 nm. Our findings suggest recurrent swap of receptors and viral pentamers as well as receptor aggregation in nanodomains. We discuss the prospects of our technique for studying virus-membrane interactions and for resolving nanoscopic dynamics of individual biological nano-objects.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    Proposed method for molecular optical imaging. Opt. Lett. 20, 237–239 (1995).

  2. 2.

    Far-field optical nanoscopy. Science 316, 1153–1158 (2007).

  3. 3.

    et al. Nanometer resolution and coherent optical dipole coupling of two individual molecules. Science 298, 385–389 (2002).

  4. 4.

    et al. Myosin V walks hand-over-hand: Single fluorophore imaging with 1.5-nm localization. Science 300, 2061–2065 (2003).

  5. 5.

    et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

  6. 6.

    , & Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).

  7. 7.

    et al. Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories. Proc. Natl. Acad. Sci. USA 104, 17370–17375 (2007).

  8. 8.

    & Illuminating single molecules in condensed matter. Science 283, 1670–1676 (1999).

  9. 9.

    Fluorescence spectroscopy of single biomolecules. Science 283, 1676–1683 (1999).

  10. 10.

    , & Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–2783 (2002).

  11. 11.

    , , , & Imaging of single molecule diffusion. Proc. Natl. Acad. Sci. USA 93, 2926–2929 (1996).

  12. 12.

    et al. Real-time single-molecule imaging of the infection pathway of an adeno-associated virus. Science 294, 1929–1932 (2001).

  13. 13.

    , , & Detection and spectroscopy of gold nanoparticle using supercontinuum white light confocal microscopy. Phys. Rev. Lett. 93, 037401 (2004).

  14. 14.

    , , , & Interferometric optical detection and tracking of very small gold nanoparticles at a water-glass interface. Opt. Express 14, 405–414 (2006).

  15. 15.

    et al. Label-free optical detection and tracking of single virions bound to their receptor in supported membrane bilayers. Nano Lett. 7, 2263–2266 (2007).

  16. 16.

    et al. Structure of Simian Virus-40 at 3.8-A resolution. Nature 354, 278–284 (1991).

  17. 17.

    et al. Gangliosides are receptors for murine polyoma virus and SV40. EMBO J. 22, 4346–4355 (2003).

  18. 18.

    , , & Structural basis of GM1 ganglioside recognition by Simian Virus 40. Proc. Natl. Acad. Sci. USA 105, 5219–5224 (2008).

  19. 19.

    , , , & Single molecule high-resolution colocalization of Cy3 and Cy5 attached to macromolecules measures intramolecular distances through time. Proc. Natl. Acad. Sci. USA 102, 1419–1423 (2005).

  20. 20.

    , , & Imaging a single quantum dot when it is dark. Nano Lett. 9, 926–929 (2009).

  21. 21.

    A new determination of the molecular dimensions. Ann. Phys. 19, 289–306 (1906).

  22. 22.

    et al. Single-particle tracking of murine polyoma virus-like particles on live cells and artificial membranes. Proc. Natl. Acad. Sci. USA 102, 15110–15115 (2005).

  23. 23.

    et al. GM1 clustering inhibits cholera toxin binding in supported phospholipid membranes. J. Am. Chem. Soc. 129, 5954–5961 (2007).

  24. 24.

    et al. Condensing and fluidizing effects of ganglioside GM1 on phospholipid films. Biophys. J. 94, 3047–3064 (2008).

  25. 25.

    et al. Gangliosides GM1 and GM3 in the living cell membrane form clusters susceptible to cholesterol depletion and chilling. Mol. Biol. Cell 18, 2112–2122 (2007).

  26. 26.

    et al. Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457, 1159–1162 (2009).

  27. 27.

    , & Interferometric detection and tracking of nanoparticles. in Nano Biophotonics (eds., Masuhara, H., Kawata, S. & Tokunaga, F.) 143–160 (Elsevier, Amsterdam, 2007).

  28. 28.

    et al. Clathrin- and caveolin-1independent endocytosis: entry of Simian Virus 40 into cells devoid of caveolae. J. Cell Biol. 168, 477–488 (2005).

  29. 29.

    et al. Paradigm shift of the plasma membrane concept from the two-dimensional continuum fluid to the partitioned fluid: High-speed single-molecule tracking of membrane molecules. Annu. Rev. Biophys. Biomol. Struct. 34, 351–378 (2005).

  30. 30.

    , & Formation of supported planar bilayers by fusion of vesicles to supported phospholipid monolayers. Biochim. Biophys. Acta 1103, 307–316 (1992).

  31. 31.

    et al. Purification and characterization of virus-like particles and pentamers by the expression of SV40 capsid proteins in insect cells. Biochim. Biophys. Acta 1290, 37–45 (1996).

Download references

Acknowledgements

We thank the Swiss Ministry of Education and Research for financial support (EU Integrated project Molecular Imaging), J. Helenius for comments, R. Mancini for providing electron micrographs of quantum dots, A. Oppenheim (Department of Hematology, Hebrew University, Hadassah Medical School and Hadassah Hospital, Jerusalem) for providing SV40 VLPs and Gunter Schwarzmann (Kekule-Institut für Organische Chemie, Universität Bonn) for NBD-GM1. A.H. thanks the Swiss National Science Foundation (SNF) for financial support.

Author information

Author notes

    • Christian Müller

    Present address: Laboratory of Organic Chemistry, ETH Zurich, Zurich, Switzerland.

Affiliations

  1. Laboratory of Physical Chemistry, Eidgenössische Technische Hochschule (ETH) Zurich, Zurich, Switzerland.

    • Philipp Kukura
    • , Helge Ewers
    • , Christian Müller
    • , Alois Renn
    •  & Vahid Sandoghdar
  2. Institute of Biochemistry, Eidgenössische Technische Hochschule (ETH) Zurich, Zurich, Switzerland.

    • Helge Ewers
    •  & Ari Helenius

Authors

  1. Search for Philipp Kukura in:

  2. Search for Helge Ewers in:

  3. Search for Christian Müller in:

  4. Search for Alois Renn in:

  5. Search for Ari Helenius in:

  6. Search for Vahid Sandoghdar in:

Contributions

P.K. designed the experimental setup, performed SV40 experiments and analyzed data. H.E. performed FRAP experiments and purified and labeled SV40. H.E. and C.M. prepared supported lipid bilayers. V.S. conceived and supervised the project in collaboration with A.H.; V.S., P.K. and H.E. wrote the manuscript; and P.K., H.E., C.M., A.R., A.H. and V.S. discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Vahid Sandoghdar.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–2

Videos

  1. 1.

    Supplementary Video 1

    Sequence of 500 consecutive iSCAT (left) and fluorescence (right) images acquired at a frame rate of 130 Hz. The frame rate has been reduced to 25 frames s−1 for clarity.

  2. 2.

    Supplementary Video 2

    Illustrative 3D rendering of a 200 frame selection from simultaneously acquired iSCAT and fluorescence trajectories.

  3. 3.

    Supplementary Video 3

    Sequential illustration of Figure 5b from the manuscript at the experimental acquisition speed of 25 frames s−1.

  4. 4.

    Supplementary Video 4

    Sequential illustration of Figure 5c from the manuscript at the experimental acquisition speed of 25 frames s−1.

  5. 5.

    Supplementary Video 5

    Sequential illustration of Figure 5d from the manuscript at the experimental acquisition speed of 25 frames s−1.

  6. 6.

    Supplementary Video 6

    Sequential illustration of Figure 5e from the manuscript at the experimental acquisition speed of 25 frames s−1.

  7. 7.

    Supplementary Video 7

    Sequential illustration of Figure 5f from the manuscript at the experimental acquisition speed of 25 frames s−1.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nmeth.1395

Further reading