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

Nature Methods volume 6, pages 923–927 (2009)Cite this article

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.

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Figure 1: iSCAT experimental approach.
Figure 2: Determination of localization accuracy and orientation.
Figure 3: iSCAT control experiments.
Figure 4: Diffusion at low (0.05 mol%) GM1 concentration.
Figure 5: Diffusion at high (1 mol%) GM1 concentration.

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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.

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Author notes

  1. Christian Müller

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

Authors and 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

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  1. Philipp Kukura
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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

Supplementary Text and Figures

Supplementary Figures 1–2 (PDF 257 kb)

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. (MOV 3496 kb)

Supplementary Video 2

Illustrative 3D rendering of a 200 frame selection from simultaneously acquired iSCAT and fluorescence trajectories. (MOV 3305 kb)

Supplementary Video 3

Sequential illustration of Figure 5b from the manuscript at the experimental acquisition speed of 25 frames s−1. (MOV 74 kb)

Supplementary Video 4

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

Supplementary Video 5

Sequential illustration of Figure 5d from the manuscript at the experimental acquisition speed of 25 frames s−1. (MOV 135 kb)

Supplementary Video 6

Sequential illustration of Figure 5e from the manuscript at the experimental acquisition speed of 25 frames s−1. (MOV 217 kb)

Supplementary Video 7

Sequential illustration of Figure 5f from the manuscript at the experimental acquisition speed of 25 frames s−1. (MOV 154 kb)

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Kukura, P., Ewers, H., Müller, C. et al. High-speed nanoscopic tracking of the position and orientation of a single virus. Nat Methods 6, 923–927 (2009). https://doi.org/10.1038/nmeth.1395

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