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

Abstract

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

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

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The authors declare no competing interests.

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Acknowledgements

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.

Competing interests

The authors declare no competing interests.

Correspondence to Vahid Sandoghdar.

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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Further reading

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.