Interferometric scattering microscopy reveals microsecond nanoscopic protein motion on a live cell membrane


Many of the biological functions of a cell are dictated by the intricate motion of proteins within its membrane over a spatial range of nanometres to tens of micrometres and time intervals of microseconds to minutes. This rich parameter space is not accessible by fluorescence microscopy, but it is within reach of interferometric scattering (iSCAT) particle tracking. However, as iSCAT is sensitive even to single unlabelled proteins, it is often accompanied by a large speckle-like background, which poses a substantial challenge for its application to cellular imaging. Here, we employ a new image processing approach to overcome this difficulty and demonstrate tracking of transmembrane epidermal growth factor receptors with nanometre precision in all three dimensions at up to microsecond speeds and for durations of tens of minutes. We provide examples of nanoscale motion and confinement in ubiquitous processes such as diffusion in the plasma membrane, transport on filopodia and rotational motion during endocytosis.

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Fig. 1: iSCAT microscopy on live cells.
Fig. 2: Diffusion on the plasma membrane.
Fig. 3: Mapping the journey of an EGFR.
Fig. 4: Confined diffusion recorded at 30,000 fps with 48 and 20 nm GNPs.
Fig. 5: Ultra-high-speed 3D tracking at 66,000 fps.
Fig. 6: Confined diffusion within a pit.

Data availability

The data that support the findings of this study are available from the corresponding author on reasonable request.

Code availability

Algorithms used in this study are available from the corresponding author on reasonable request.


  1. 1.

    Weisenburger, S. & Sandoghdar, V. Light microscopy: an ongoing contemporary revolution. Contemp. Phys. 56, 123–143 (2015).

    ADS  Article  Google Scholar 

  2. 2.

    De Brabander, M., Nuydens, R., Geerts, H. & Hopkins, C. R. Dynamic behavior of the transferrin receptor followed in living epidermoid carcinoma (A431) cells with nanovid microscopy. Cell Motil. Cytoskeleton 9, 30–47 (1988).

    Article  Google Scholar 

  3. 3.

    Kusumi, A., Sako, Y. & Yamamoto, M. Confined lateral diffusion of membrane-receptors as studied by single-particle tracking (nanovid microscopy). Effects of calcium-induced differentiation in cultured epithelial-cells. Biophys. J. 65, 2021–2040 (1993).

    ADS  Article  Google Scholar 

  4. 4.

    Schultz, S., Smith, D. R., Mock, J. J. & Schultz, D. A. Single-target molecule detection with nonbleaching multicolor optical immunolabels. Proc. Natl Acad. Sci. USA 97, 996–1001 (2000).

    ADS  Article  Google Scholar 

  5. 5.

    Nan, X., Sims, P. A. & Xie, X. S. Organelle tracking in a living cell with microsecond time resolution and nanometer spatial precision. ChemPhysChem 9, 707–712 (2008).

    Article  Google Scholar 

  6. 6.

    Fujiwara, T., Ritchie, K., Murakoshi, H., Jacobson, K. & Kusumi, A. Phospholipids undergo hop diffusion in compartmentalized cell membrane. J. Cell Bio. 157, 1071–1081 (2002).

    Article  Google Scholar 

  7. 7.

    Lindfors, K., Kalkbrenner, T., Stoller, P. & Sandoghdar, V. Detection and spectroscopy of gold nanoparticles using supercontinuum white light confocal microscopy. Phys. Rev. Lett. 93, 037401 (2004).

    ADS  Article  Google Scholar 

  8. 8.

    Jacobsen, V., Stoller, P., Brunner, C., Vogel, V. & Sandoghdar, V. Interferometric optical detection and tracking of very small gold nanoparticles at a water-glass interface. Opt Express 14, 405–414 (2006).

    ADS  Article  Google Scholar 

  9. 9.

    Kukura, P. et al. High-speed nanoscopic tracking of the position and orientation of a single virus. Nat. Methods 6, 923–927 (2009).

    Article  Google Scholar 

  10. 10.

    Krall, J. A., Beyer, E. M. & MacBeath, G. High- and low-affinity epidermal growth factor receptor-ligand interactions activate distinct signaling pathways. PLoS ONE 6, e15945 (2011).

    ADS  Article  Google Scholar 

  11. 11.

    Tomas, A., Futter, C. E. & Eden, E. R. EGF receptor trafficking: consequences for signaling and cancer. Trends Cell Biol. 24, 26–34 (2014).

    Article  Google Scholar 

  12. 12.

    Piliarik, M. & Sandoghdar, V. Direct optical sensing of single unlabelled proteins and super-resolution imaging of their binding sites. Nat. Commun. 5, 4495 (2014).

    ADS  Article  Google Scholar 

  13. 13.

    Ortega Arroyo, J. et al. Label-free, all-optical detection, imaging, and tracking of a single protein. Nano Lett. 14, 2065–2070 (2014).

    ADS  Article  Google Scholar 

  14. 14.

    McDonald, M. P. et al. Visualizing single-cell secretion dynamics with single-protein sensitivity. Nano Lett. 18, 513–519 (2018).

    ADS  Article  Google Scholar 

  15. 15.

    Hsieh, C.-L., Spindler, S., Ehrig, J. & Sandoghdar, V. Tracking single particles on supported lipid membranes: multimobility diffusion and nanoscopic confinement. J. Phys. Chem. B 118, 1545–1554 (2014).

    Article  Google Scholar 

  16. 16.

    Spillane, K. M. et al. High-speed single-particle tracking of GM1 in model membranes reveals anomalous diffusion due to interleaflet coupling and molecular pinning. Nano Lett. 14, 5390–5397 (2014).

    ADS  Article  Google Scholar 

  17. 17.

    Wu, H.-M., Lin, Y.-H., Yen, T.-C. & Hsieh, C.-L. Nanoscopic substructures of raft-mimetic liquid-ordered membrane domains revealed by high-speed single-particle tracking. Sci. Rep. 6, 20542 (2016).

    ADS  Article  Google Scholar 

  18. 18.

    Spindler, S. et al. Visualization of lipids and proteins at high spatial and temporal resolution via interferometric scattering (iSCAT) microscopy. J. Phys. D Appl. Phys. 49, 274002 (2016).

    Article  Google Scholar 

  19. 19.

    Park, J.-S. et al. Label-free and live cell imaging by interferometric scattering microscopy. Chem. Sci. 9, 2690–2697 (2018).

    Article  Google Scholar 

  20. 20.

    Reina, F. et al. Complementary studies of lipid membrane dynamics using iSCAT and super-resolved fluorescence correlation spectroscopy. J. Phys. D Appl. Phys. 51, 235401 (2018).

    ADS  Article  Google Scholar 

  21. 21.

    Etoc, F. et al. Diffusion of nanosized objects in mammalian cells. Nat. Mater. 17, 740–746 (2018).

    ADS  Article  Google Scholar 

  22. 22.

    Huang, Y.-F. et al. Coherent brightfield microscopy provides the spatiotemporal resolution to study early stage viral infection in live cells. ACS Nano 11, 2575–2585 (2017).

    Article  Google Scholar 

  23. 23.

    de Wit, G., Albrecht, D., Ewers, H. & Kukura, P. Revealing compartmentalized diffusion in living cells with interferometric scattering microscopy. Biophys. J. 114, 2945–2950 (2018).

    Article  Google Scholar 

  24. 24.

    Krishnan, M., Mojarad, N., Kukura, P. & Sandoghdar, V. Geometry-induced electrostatic trapping of nanometric objects in a fluid. Nature 467, 692–695 (2010).

    ADS  Article  Google Scholar 

  25. 25.

    Manzo, C. & Garcia-Parajo, M. F. A review of progress in single particle tracking: from methods to biophysical insights. Rep. Prog. Phys. 78, 124601 (2015).

    ADS  Article  Google Scholar 

  26. 26.

    Kusumi, A., Tsunoyama, T. A., Hirosawa, K. M., Kasai, R. S. & Fujiwara, T. K. Tracking single molecules at work in living cells. Nat. Chem. Biol. 10, 524–532 (2014).

    Article  Google Scholar 

  27. 27.

    Martin, D. S., Forstner, M. B. & Käs, J. A. Apparent subdiffusion inherent to single particle tracking. Biophys. J. 83, 2109–2117 (2002).

    ADS  Article  Google Scholar 

  28. 28.

    Saxton, M. J. A biological interpretation of transient anomalous subdiffusion. I. Qualitative model. Biophys. J. 92, 1178–1191 (2007).

    ADS  Article  Google Scholar 

  29. 29.

    Simons, K. & Sampaio, J. L. Membrane organization and lipid rafts. Cold Spring Harb. Perspect. Biol. 3, a004697 (2011).

    Article  Google Scholar 

  30. 30.

    Simson, R., Sheets, E. D. & Jacobson, K. Detection of temporary lateral confinement of membrane proteins using single-particle tracking analysis. Biophys. J. 69, 989–993 (1995).

    ADS  Article  Google Scholar 

  31. 31.

    Kalay, Z., Parris, P. E. & Kenkre, V. M. Effects of disorder in location and size of fence barriers on molecular motion in cell membranes. J. Phys. Condens. Mat. 20, 245105 (2008).

    ADS  Article  Google Scholar 

  32. 32.

    Wieser, S. & Schütz, G. J. Tracking single molecules in the live cell plasma membrane-do’s and don’t’s. Methods 46, 131–140 (2008).

    Article  Google Scholar 

  33. 33.

    Saxton, M. J. & Jacobson, K. Single-particle tracking: applications to membrane dynamics. Annu. Rev. Bioph. Biom. 26, 373–399 (1997).

    Article  Google Scholar 

  34. 34.

    Weeks, E. R. & Weitz, D. A. Properties of cage rearrangements observed near the colloidal glass transition. Phys. Rev. Lett. 89, 095704 (2002).

    ADS  Article  Google Scholar 

  35. 35.

    Mattila, P. K. & Lappalainen, P. Filopodia: molecular architecture and cellular functions. Nat. Rev. Mol. Cell Biol. 9, 446–454 (2008).

    Article  Google Scholar 

  36. 36.

    Kornberg, T. B. Distributing signaling proteins in space and time: the province of cytonemes. Curr. Opin. Genet. Dev. 45, 22–27 (2017).

    Article  Google Scholar 

  37. 37.

    Lidke, D. S. et al. Quantum dot ligands provide new insights into erbB/HER receptor-mediated signal transduction. Nat. Biotechnol. 22, 198–203 (2004).

    ADS  Article  Google Scholar 

  38. 38.

    Arndt-Jovin, D. J., Botelho, M. G. & Jovin, T. M. Structure-function relationships of ErbB RTKs in the plasma membrane of living cells. Cold Spring Harb. Perspect. Biol. 6, a008961 (2014).

    Article  Google Scholar 

  39. 39.

    Michalet, X. Mean square displacement analysis of single-particle trajectories with localization error: Brownian motion in an isotropic medium. Phys. Rev. E 82, 041914 (2010).

    ADS  MathSciNet  Article  Google Scholar 

  40. 40.

    Liu, Y.-L. et al. Segmentation of 3D trajectories acquired by TSUNAMI microscope: an application to EGFR trafficking. Biophy. J. 111, 2214–2227 (2016).

    ADS  Article  Google Scholar 

  41. 41.

    Ritchie, K. et al. Detection of non-Brownian diffusion in the cell membrane in single molecule tracking. Biophys. J. 88, 2266–2277 (2005).

    ADS  Article  Google Scholar 

  42. 42.

    Kirchhausen, T. Imaging endocytic clathrin structures in living cells. Trends Cell Biol. 19, 596–605 (2009).

    Article  Google Scholar 

  43. 43.

    Grove, J. et al. Flat clathrin lattices: stable features of the plasma membrane. Mol. Biol. Cell 25, 3581–3594 (2014).

    Article  Google Scholar 

  44. 44.

    Sigismund, S. et al. Clathrin-mediated internalization is essential for sustained EGFR signaling but dispensable for degradation. Dev. Cell 15, 209–219 (2008).

    Article  Google Scholar 

  45. 45.

    Ruthardt, N., Lamb, D. C. & Bräuchle, C. Single-particle tracking as a quantitative microscopy-based approach to unravel cell entry mechanisms of viruses and pharmaceutical nanoparticles. Mol. Ther. 19, 1199–1211 (2011).

    Article  Google Scholar 

  46. 46.

    Brodsky, F. M. Diversity of clathrin function: new tricks for an old protein. Annu. Rev. Cell Dev. Biol. 28, 309–336 (2012).

    MathSciNet  Article  Google Scholar 

  47. 47.

    McMahon, H. T. & Boucrot, E. Molecular mechanism and physiological functions of clathrin-mediated endocytosis. Nat. Rev. Mol. Cell Biol. 12, 517–533 (2011).

    Article  Google Scholar 

  48. 48.

    Roux, A., Uyhazi, K., Frost, A. & De Camilli, P. GTP-dependent twisting of dynamin implicates constriction and tension in membrane fission. Nature 441, 528–531 (2006).

    ADS  Article  Google Scholar 

  49. 49.

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

    ADS  Article  Google Scholar 

  50. 50.

    Wäldchen, S., Lehmann, J., Klein, T., van de Linde, S. & Sauer, M. Light-induced cell damage in live-cell super-resolution microscopy. Sci. Rep. 5, 15348 (2015).

    ADS  Article  Google Scholar 

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This project was funded by an Alexander von Humboldt professorship, the Max Planck Society and the Research and Training Grant 1962 (‘Dynamic Interactions at Biological Membranes’) of the German Research Foundation. R.W.T. acknowledges an Alexander von Humboldt fellowship. V.R. and A.S. were also supported by a grant from the German Research Foundation (grant no. SCHA965/9-1). We thank S. Ihloff for support in cell culturing, C. Obermeier for preparing ultra-thin sections (TEM), B. Schmid (Optical Imaging Center Erlangen) for support in co-localization analyses and V. Zaburdaev for insightful discussions regarding statistical analysis of diffusion.

Author information




R.W.T. made iSCAT measurements and performed data analysis. R.G.M. performed the quantitative analysis of iSCAT images and trajectories. V.R. prepared TEM samples and carried out immunofluorescence and western blot experiments. A.G. performed electron microscopy. A.S. supervised biological preparation and data interpretation. V.S. conceived and supervised the project. V.S., R.W.T. and A.S. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Vahid Sandoghdar.

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Supplementary information

Supplementary Information

This file contains more information about the work and Supplementary Figures 1–7.

Reporting Summary

Supplementary Video 1

Raw video of an EGFR–GNP diffusing on a HeLa cell membrane.

Supplementary Video 2

Diffusion on a filopodium.

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Taylor, R.W., Mahmoodabadi, R.G., Rauschenberger, V. et al. Interferometric scattering microscopy reveals microsecond nanoscopic protein motion on a live cell membrane. Nat. Photonics 13, 480–487 (2019).

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