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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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.
Supplementary Figures 1–2 (PDF 257 kb)
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)
Illustrative 3D rendering of a 200 frame selection from simultaneously acquired iSCAT and fluorescence trajectories. (MOV 3305 kb)
Sequential illustration of Figure 5b from the manuscript at the experimental acquisition speed of 25 frames s−1. (MOV 74 kb)
Sequential illustration of Figure 5c from the manuscript at the experimental acquisition speed of 25 frames s−1. (MOV 25 kb)
Sequential illustration of Figure 5d from the manuscript at the experimental acquisition speed of 25 frames s−1. (MOV 135 kb)
Sequential illustration of Figure 5e from the manuscript at the experimental acquisition speed of 25 frames s−1. (MOV 217 kb)
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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