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Direct observation of the nanoscale dynamics of membrane lipids in a living cell


Cholesterol-mediated lipid interactions are thought to have a functional role in many membrane-associated processes such as signalling events1,2,3,4,5. Although several experiments indicate their existence, lipid nanodomains (‘rafts’) remain controversial owing to the lack of suitable detection techniques in living cells4,6,7,8,9. The controversy is reflected in their putative size of 5–200 nm, spanning the range between the extent of a protein complex and the resolution limit of optical microscopy. Here we demonstrate the ability of stimulated emission depletion (STED) far-field fluorescence nanoscopy10 to detect single diffusing (lipid) molecules in nanosized areas in the plasma membrane of living cells. Tuning of the probed area to spot sizes 70-fold below the diffraction barrier reveals that unlike phosphoglycerolipids, sphingolipids and glycosylphosphatidylinositol-anchored proteins are transiently (10–20 ms) trapped in cholesterol-mediated molecular complexes dwelling within <20-nm diameter areas. The non-invasive optical recording of molecular time traces and fluctuation data in tunable nanoscale domains is a powerful new approach to study the dynamics of biomolecules in living cells.

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Figure 1: STED microscopy time traces of single-molecule diffusion in live cell plasma membrane.
Figure 2: FCS of Atto647N-labelled phosphoethanolamine and sphingomyelin plasma membrane diffusion.
Figure 3: Molecular transit through nanoscale areas in live cell plasma membrane.


  1. Simons, K. & Ikonen, E. Functional rafts in cell membranes. Nature 387, 569–572 (1997)

    Article  ADS  CAS  Google Scholar 

  2. Brown, D. A. & London, E. Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J. Biol. Chem. 275, 17221–17224 (2000)

    Article  CAS  Google Scholar 

  3. Fielding, C. J. Lipid Rafts and Caveolae (Wiley-VCH, 2006)

    Book  Google Scholar 

  4. Jacobson, K., Mouritsen, O. G. & Anderson, G. W. Lipid rafts: at a crossroad between cell biology and physics. Nature Cell Biol. 9, 7–14 (2007)

    Article  ADS  CAS  Google Scholar 

  5. Hanzal-Bayer, M. F. & Hancock, J. F. Lipid rafts and membrane traffic. FEBS Lett. 581, 2098–2104 (2007)

    Article  CAS  Google Scholar 

  6. Munro, S. Lipid rafts: elusive or illusive? Cell 115, 377–388 (2003)

    Article  CAS  Google Scholar 

  7. Lommerse, P. H. M., Spaink, H. P. & Schmidt, T. In vivo plasma membrane organization: results of biophysical approaches. Biochim. Biophys. Acta 1664, 119–131 (2004)

    Article  CAS  Google Scholar 

  8. Hancock, J. F. Lipid rafts: contentious only from simplistic standpoints. Nature Rev. Mol. Cell Biol. 7, 456–462 (2006)

    Article  CAS  Google Scholar 

  9. Shaw, A. S. Lipid rafts: now you see them, now you don’t. Nature Immunol. 7, 1139–1142 (2006)

    Article  CAS  Google Scholar 

  10. Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated emission depletion microscopy. Opt. Lett. 19, 780–782 (1994)

    Article  ADS  CAS  Google Scholar 

  11. Pike, L. J. Rafts defined: a report on the Keystone symposium on lipid rafts and cell function. J. Lipid Res. 47, 1597–1598 (2006)

    Article  CAS  Google Scholar 

  12. Fujita, A. 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)

    Article  CAS  Google Scholar 

  13. Binnig, G., Quate, C. F. & Gerber, C. Atomic force microscope. Phys. Rev. Lett. 56, 930–933 (1986)

    Article  ADS  CAS  Google Scholar 

  14. Pohl, D. W., Denk, W. & Lanz, M. Optical stethoscopy: Image recording with resolution λ/20. Appl. Phys. Lett. 44, 651–653 (1984)

    Article  ADS  Google Scholar 

  15. Zacharias, D. A., Violin, J. D., Newton, A. C. & Tsien, R. Y. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002)

    Article  ADS  CAS  Google Scholar 

  16. Saxton, M. J. & Jacobson, K. Single particle tracking: applications to membrane dynamics. Annu. Rev. Biophys. Biomol. Struct. 26, 373–399 (1997)

    Article  CAS  Google Scholar 

  17. Schütz, G. J., Kada, G., Pastushenko, V. P. & Schindler, H. Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy. EMBO J. 19, 892–901 (2000)

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  19. Yechiel, E. & Edidin, M. Micrometer-scale domains in fibroblast plasma membranes. J. Cell Biol. 105, 755–760 (1987)

    Article  CAS  Google Scholar 

  20. Feder, T. J., Brust-Mascher, I., Slattery, J. P., Baird, B. A. & Webb, W. W. Constrainted diffusion or immobile fraction on cell surfaces: a new interpretation. Biophys. J. 70, 2767–2773 (1996)

    Article  ADS  CAS  Google Scholar 

  21. Fahey, P. F. et al. Lateral diffusion in planar lipid bilayers. Science 195, 305–306 (1977)

    Article  ADS  CAS  Google Scholar 

  22. Schwille, P., Korlach, J. & Webb, W. W. Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes. Cytometry 36, 176–182 (1999)

    Article  CAS  Google Scholar 

  23. Wawrezinieck, L., Rigneault, H., Marguet, D. & Lenne, P.-F. Fluorescence correlation spectroscopy: diffusion laws to probe the submicron cell membrane organization. Biophys. J. 89, 4029–4042 (2005)

    Article  CAS  Google Scholar 

  24. Wenger, J. et al. Diffusion analysis within single nanometric apertures reveals the ultrafine cell membrane organization. Biophys. J. 92, 913–919 (2007)

    Article  ADS  CAS  Google Scholar 

  25. Willig, K. I., Rizzoli, S. O., Westphal, V., Jahn, R. & Hell, S. W. STED-microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature 440, 935–939 (2006)

    Article  ADS  CAS  Google Scholar 

  26. Hell, S. W. Far-field optical nanoscopy. Science 316, 1153–1158 (2007)

    Article  ADS  CAS  Google Scholar 

  27. Magde, D., Elson, E. L. & Webb, W. W. Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy. Phys. Rev. Lett. 29, 705–708 (1972)

    Article  ADS  CAS  Google Scholar 

  28. Osborn, M., Franke, W. W. & Weber, K. Visualization of a system of filaments 7–10 nm thick in cultured cells of an epithelioid line (PtK2) by immunofluorescence microscopy. Proc. Natl Acad. Sci. USA 74, 2490–2494 (1977)

    Article  ADS  CAS  Google Scholar 

  29. Martin, O. C. & Pagano, R. C. Internalization and sorting of a fluorescent analogue of glucosylceramide to the Golgi apparatus of human skin fibroblasts: utilization of endocytic and nonendocytic transport mechanisms. J. Cell Biol. 125, 769–781 (1994)

    Article  CAS  Google Scholar 

  30. Schwarzmann, G., Hofmann, P., Pütz, U. & Albrecht, B. Demonstration of direct glycosylation of nondegradable glucosylceramide analogs in cultured cells. J. Biol. Chem. 270, 21271–21276 (1995)

    Article  CAS  Google Scholar 

  31. Keller, J., Schönle, A. & Hell, S. W. Efficient fluorescence inhibition patterns for RESOLFT microscopy. Opt. Express 15, 3361–3371 (2007)

    Article  ADS  Google Scholar 

  32. Willig, K. I. et al. Nanoscale resolution in GFP-based microscopy. Nature Methods 3, 721–723 (2006)

    Article  CAS  Google Scholar 

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We thank J. Jethwa, B. Rankin and M. Hilbert for critical reading, K. Willig for help with the setup, T. Lang, R. Wagner and H. Rigneault for valuable discussions, R. Machinek and H. Frauendorf for recording the NMR and mass spectra, and S. Yan for help with the synthesis.

Author Contributions C.R. and B.H. performed experiments, C.E. and C.R. analysed data, R.M. prepared samples and performed washing experiments, G.S., K.S., S.P. and V.N.B. synthesized fluorescently labelled lipids and performed chromatography, C.v.M., A.S., C.R. and C.E. realized and analysed simulated data, C.E. and S.W.H. designed experiments and wrote the paper. All authors discussed the results and commented on the manuscript.

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Correspondence to Christian Eggeling or Stefan W. Hell.

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

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