Single-Molecule Analysis of Biomembranes

  • Thomas Schmidt
  • Gerhard J. Schütz


Biomembranes are more than just a cell’s envelope – as the interface to the surrounding of a cell they carry key signalling functions. Consequentially, membranes are highly complex organelles: they host about thousand different types of lipids and about half of the proteome, whose interaction has to be orchestrated appropriately for the various signalling purposes. In particular, knowledge on the nanoscopic organization of the plasma membrane appears critical for understanding the regulation of interactions between membrane proteins. The high localization precision of ∼20 nm combined with a high time resolution of ∼1 ms made single molecule tracking an excellent technology to obtain insights into membrane nanostructures, even in a live cell context. In this chapter, we will highlight concepts to achieve superresolution by single molecule imaging, summarize tools for data analysis, and review applications on artificial and live cell membranes.


Lipid Raft Point Spread Function Point Light Source Localization Precision Macroscopic Phase Separation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Singer, S. J., and G. L. Nicolson. 1972. The fluid mosaic model of the structure of cell membranes. Science 175:720–731.ADSCrossRefGoogle Scholar
  2. 2.
    Lipowsky, R., and E. Sackmann, editors. 1995. Structure and dynamics of membranes. Amsterdam: Elsevier.Google Scholar
  3. 3.
    Vereb, G., J. Szollosi, J. Matko, P. Nagy, T. Farkas, L. Vigh, L. Matyus, T. A. Waldmann, and S. Damjanovich. 2003. Dynamic, yet structured: the cell membrane three decades after the Singer-Nicolson model. Proc Natl Acad Sci USA 100:8053–8058.ADSCrossRefGoogle Scholar
  4. 4.
    Jacobson, K., O. G. Mouritsen, and R. G. Anderson. 2007. Lipid rafts: at a crossroad between cell biology and physics. Nat Cell Biol 9:7–14.CrossRefGoogle Scholar
  5. 5.
    Kusumi, A., C. Nakada, K. Ritchie, K. Murase, K. Suzuki, H. Murakoshi, R. S. Kasai, J. Kondo, and T. Fujiwara. 2005. Paradigm shift of the plasma membrane concept from the two-dimensional continuum fluid to the partitioned fluid: high-speed single-molecule tracking of membrane molecules. Annu Rev Biophys Biomol Struct 34:351–378.CrossRefGoogle Scholar
  6. 6.
    Lenne, P. F., L. Wawrezinieck, F. Conchonaud, O. Wurtz, A. Boned, X. J. Guo, H. Rigneault, H. T. He, and D. Marguet. 2006. Dynamic molecular confinement in the plasma membrane by microdomains and the cytoskeleton meshwork. EMBO J 25:3245–3256.CrossRefGoogle Scholar
  7. 7.
    Anderson, R. G., and K. Jacobson. 2002. A role for lipid shells in targeting proteins to caveolae, rafts, and other lipid domains. Science 296:1821–1825.ADSCrossRefGoogle Scholar
  8. 8.
    Fujiwara, T., K. Ritchie, H. Murakoshi, K. Jacobson, and A. Kusumi. 2002. Phospholipids undergo hop diffusion in compartmentalized cell membrane. J Cell Biol 157:1071–1081.CrossRefGoogle Scholar
  9. 9.
    Chen, Y., W. R. Thelin, B. Yang, S. L. Milgram, and K. Jacobson. 2006. Transient anchorage of cross-linked glycosyl-phosphatidylinositol-anchored proteins depends on cholesterol, Src family kinases, caveolin, and phosphoinositides. J Cell Biol 175:169–178.CrossRefGoogle Scholar
  10. 10.
    Suzuki, K. G., T. K. Fujiwara, F. Sanematsu, R. Iino, M. Edidin, and A. Kusumi. 2007. GPI-anchored receptor clusters transiently recruit Lyn and G{alpha} for temporary cluster immobilization and Lyn activation: single-molecule tracking study 1. J Cell Biol 177: 717–730.CrossRefGoogle Scholar
  11. 11.
    Demond, A. L., K. D. Mossman, T. Starr, M. L. Dustin, and J. T. Groves. 2008. T cell receptor microcluster transport through molecular mazes reveals mechanism of translocation. Biophys J 94:3286–3292.Google Scholar
  12. 12.
    Sako, Y., A. Nagafuchi, S. Tsukita, M. Takeichi, and A. Kusumi. 1998. Cytoplasmic regulation of the movement of E-cadherin on the free cell surface as studied by optical tweezers and single particle tracking: corralling and tethering by the membrane skeleton. J Cell Biol 140:1227–1240.CrossRefGoogle Scholar
  13. 13.
    Saxton, M. J. 1994. Anomalous diffusion due to obstacles: a Monte Carlo study. Biophys J 66:394–401.CrossRefGoogle Scholar
  14. 14.
    Marguet, D., P. F. Lenne, H. Rigneault, and H. T. He. 2006. Dynamics in the plasma membrane: how to combine fluidity and order. EMBO J 25:3446–3457.CrossRefGoogle Scholar
  15. 15.
    Simons, K., and E. Ikonen. 1997. Functional rafts in cell membranes. Nature 387:569–572.ADSCrossRefGoogle Scholar
  16. 16.
    Barak, L. S., and W. W. Webb. 1981. Fluorescent low density lipoprotein for observation of dynamics of individual receptor complexes on cultured human fibroblasts. J Cell Biol 90:595–604.CrossRefGoogle Scholar
  17. 17.
    Kusumi, A., Y. Sako, and M. Yamamoto. 1993. 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.CrossRefGoogle Scholar
  18. 18.
    Saxton, M. J., and K. Jacobson. 1997. Single-particle tracking: applications to membrane dynamics. Annu Rev Biophys Biomol Struct 26:373–399.CrossRefGoogle Scholar
  19. 19.
    Lee, G. M., A. Ishihara, and K. A. Jacobson. 1991. Direct observation of Brownian motion of lipids in a membrane. Proc Natl Acad Sci USA 88:6274–6278.ADSCrossRefGoogle Scholar
  20. 20.
    Wilson, K. M., I. E. Morrison, P. R. Smith, N. Fernandez, and R. J. Cherry. 1996. Single particle tracking of cell-surface HLA-DR molecules using R-phycoerythrin labeled monoclonal antibodies and fluorescence digital imaging. J Cell Sci 109 (Pt 8):2101–2109.Google Scholar
  21. 21.
    Kao, H. P., and A. S. Verkman. 1994. Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position. Biophys J 67:1291–1300.CrossRefGoogle Scholar
  22. 22.
    Felsenfeld, D. P., D. Choquet, and M. P. Sheetz. 1996. Ligand binding regulates the directed movement of beta1 integrins on fibroblasts. Nature 383:438–440.ADSCrossRefGoogle Scholar
  23. 23.
    Anderson, C. M., G. N. Georgiou, I. E. Morrison, G. V. Stevenson, and R. J. Cherry. 1992. Tracking of cell surface receptors by fluorescence digital imaging microscopy using a charge-coupled device camera. Low-density lipoprotein and influenza virus receptor mobility at 4 degrees C. J Cell Sci 101(Pt 2): 415–425.Google Scholar
  24. 24.
    Fein, M., J. Unkeless, F. Y. Chuang, M. Sassaroli, R. da Costa, H. Vaananen, and J. Eisinger. 1993. Lateral mobility of lipid analogues and GPI-anchored proteins in supported bilayers determined by fluorescent bead tracking. J Membr Biol 135:83–92.Google Scholar
  25. 25.
    Geerts, H., M. De Brabander, R. Nuydens, S. Geuens, M. Moeremans, J. De Mey, and P. Hollenbeck. 1987. Nanovid tracking: a new automatic method for the study of mobility in living cells based on colloidal gold and video microscopy. Biophys J 52: 775–782.CrossRefGoogle Scholar
  26. 26.
    Thompson, R. E., D. R. Larson, and W. W. Webb. 2002. Precise nanometer localization analysis for individual fluorescent probes. Biophys J 82:2775–2783.CrossRefGoogle Scholar
  27. 27.
    Kuno, M., D. P. Fromm, H. F. Hamann, A. Gallagher, and D. J. Nesbitt. 1999. Nonexponential “blinking” kinetics of single CdSe quantum dots: a universal power law behavior. J Chem Phys 112:3117–3120.ADSCrossRefGoogle Scholar
  28. 28.
    Schmidt, T., G. J. Schütz, W. Baumgartner, H. J. Gruber, and H. Schindler. 1995. Characterization of photophysics and mobility of single molecules in a fluid lipid membrane. J Phys Chem 99: 17662–17668.CrossRefGoogle Scholar
  29. 29.
    Schmidt, T., G. J. Schütz, W. Baumgartner, H. J. Gruber, and H. Schindler. 1996. Imaging of single molecule diffusion. Proc Natl Acad Sci USA 93:2926–2929.ADSCrossRefGoogle Scholar
  30. 30.
    Funatsu, T., Y. Harada, M. Tokunaga, K. Saito, and T. Yanagida. 1995. Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous-solution. Nature 374:555–559.ADSCrossRefGoogle Scholar
  31. 31.
    Sase, I., H. Miyata, J. E. Corrie, J. S. Craik, and K. Kinosita, Jr. 1995. Real time imaging of single fluorophores on moving actin with an epifluorescence microscope. Biophys J 69: 323–328.CrossRefGoogle Scholar
  32. 32.
    Schütz, G. J., H. Schindler, and T. Schmidt. 1997. Single-molecule microscopy on model membranes reveals anomalous diffusion. Biophys J 73:1073–1080.CrossRefGoogle Scholar
  33. 33.
    Schütz, G. J., W. Trabesinger, and T. Schmidt. 1998. Direct observation of ligand colocalization on individual receptor molecules. Biophys J 74:2223–2226.CrossRefGoogle Scholar
  34. 34.
    Sonnleitner, A., G. J. Schutz, and T. Schmidt. 1999. Free Brownian motion of individual lipid molecules in biomembranes. Biophys J 77:2638–2642.CrossRefGoogle Scholar
  35. 35.
    Harms, G. S., M. Sonnleitner, G. J. Schütz, H. J. Gruber, and T. Schmidt. 1999. Single-molecule anisotropy imaging. Biophys J 77:2864–2870.CrossRefGoogle Scholar
  36. 36.
    Ke, P. C., and C. A. Naumann. 2001. Hindered diffusion in polymer-tethered phosopholipid monolayers at the air–water interface: a single molecule fluorescence imaging study. Langmuir 17:5076–5081.CrossRefGoogle Scholar
  37. 37.
    Ke, P. C., and C. A. Naumann. 2001. Single molecule fluorescence imaging of phospholipid monolayers at the air–water interface. Langmuir 17:3727–3733.CrossRefGoogle Scholar
  38. 38.
    Deverall, M. A., E. Gindl, E. K. Sinner, H. Besir, J. Ruehe, M. J. Saxton, and C. A. Naumann. 2005. Membrane lateral mobility obstructed by polymer-tethered lipids studied at the single molecule level. Biophys J 88: 1875–1886.CrossRefGoogle Scholar
  39. 39.
    Kiessling, V., J. M. Crane, and L. K. Tamm. 2006. Transbilayer effects of raft-like lipid domains in asymmetric planar bilayers measured by single molecule tracking. Biophys J 91:3313–3326.CrossRefGoogle Scholar
  40. 40.
    Schütz, G. J., G. Kada, V. P. Pastushenko, and H. Schindler. 2000. Properties of lipid microdomains in a muscle cell membrane visualized by single molecule microscopy. EMBO J 19:892–901.CrossRefGoogle Scholar
  41. 41.
    Schütz, G. J., V. P. Pastushenko, H. J. Gruber, H.-G. Knaus, B. Pragl, and H. Schindler. 2000. 3D Imaging of individual ion channels in live cells at 40 nm resolution. Single Mol. 1:25–31.ADSCrossRefGoogle Scholar
  42. 42.
    Wieser, S., M. Moertelmaier, E. Fuertbauer, H. Stockinger, and G. J. Schutz. 2007. (Un)Confined diffusion of CD59 in the plasma membrane determined by high-resolution single molecule microscopy. Biophys J 92: 3719–3728.CrossRefGoogle Scholar
  43. 43.
    Drbal, K., M. Moertelmaier, C. Holzhauser, A. Muhammad, E. Fuertbauer, S. Howorka, M. Hinterberger, H. Stockinger, and G. J. Schutz. 2007. Single-molecule microscopy reveals heterogeneous dynamics of lipid raft components upon TCR engagement. Int Immunol 19:675–684.CrossRefGoogle Scholar
  44. 44.
    Wieser, S., G. J. Schutz, M. E. Cooper, and H. Stockinger. 2007. Single molecule diffusion analysis on cellular nanotubules: implications on plasma membrane structure below the diffraction limit. Appl Phys Lett 91:233901.ADSCrossRefGoogle Scholar
  45. 45.
    Lommerse, P. H., K. Vastenhoud, N. J. Pirinen, A. I. Magee, H. P. Spaink, and T. Schmidt. 2006. Single-molecule diffusion reveals similar mobility for the Lck, H-ras, and K-ras membrane anchors. Biophys J 91:1090–1097.CrossRefGoogle Scholar
  46. 46.
    Lommerse, P. H., B. E. Snaar-Jagalska, H. P. Spaink, and T. Schmidt. 2005. Single-molecule diffusion measurements of H-Ras at the plasma membrane of live cells reveal microdomain localization upon activation. J Cell Sci 118:1799–1809.CrossRefGoogle Scholar
  47. 47.
    Lommerse, P. H., G. A. Blab, L. Cognet, G. S. Harms, B. E. Snaar-Jagalska, H. P. Spaink, and T. Schmidt. 2004. Single-molecule imaging of the H-Ras membrane-anchor reveals domains in the cytoplasmic leaflet of the cell membrane. Biophys J 86:609–616.CrossRefGoogle Scholar
  48. 48.
    Harms, G. S., L. Cognet, P. H. Lommerse, G. A. Blab, H. Kahr, R. Gamsjager, H. P. Spaink, N. M. Soldatov, C. Romanin, and T. Schmidt. 2001. Single-molecule imaging of l-type Ca(2+) channels in live cells. Biophys J 81:2639–2646.CrossRefGoogle Scholar
  49. 49.
    Vrljic, M., S. Y. Nishimura, S. Brasselet, W. E. Moerner, and H. M. McConnell. 2002. Translational diffusion of individual class II MHC membrane proteins in cells. Biophys J 83:2681–2692.CrossRefGoogle Scholar
  50. 50.
    Vrljic, M., S. Y. Nishimura, W. E. Moerner, and H. M. McConnell. 2005. Cholesterol depletion suppresses the translational diffusion of class II major histocompatibility complex proteins in the plasma membrane. Biophys J 88:334–347.CrossRefGoogle Scholar
  51. 51.
    Nishimura, S. Y., M. Vrljic, L. O. Klein, H. M. McConnell, and W. E. Moerner. 2006. Cholesterol depletion induces solid-like regions in the plasma membrane. Biophys J 90: 927–938.CrossRefGoogle Scholar
  52. 52.
    Umemura, Y. M., M. Vrljic, S. Y. Nishimura, T. K. Fujiwara, K. G. Suzuki, and A. Kusumi. 2008. Both MHC class II and its GPI-anchored form undergo hop diffusion as observed by single-molecule tracking. Biophys J 95:435–450.Google Scholar
  53. 53.
    Murase, K., T. Fujiwara, Y. Umemura, K. Suzuki, R. Iino, H. Yamashita, M. Saito, H. Murakoshi, K. Ritchie, and A. Kusumi. 2004. Ultrafine membrane compartments for molecular diffusion as revealed by single molecule techniques. Biophys J 86:4075–4093.CrossRefGoogle Scholar
  54. 54.
    Douglass, A. D., and R. D. Vale. 2005. Single-molecule microscopy reveals plasma membrane microdomains created by protein-protein networks that exclude or trap signaling molecules in T cells. Cell 121:937–950.CrossRefGoogle Scholar
  55. 55.
    Hess, S. T., T. J. Gould, M. V. Gudheti, S. A. Maas, K. D. Mills, and J. Zimmerberg. 2007. Dynamic clustered distribution of hemagglutinin resolved at 40 nm in living cell membranes discriminates between raft theories. Proc Natl Acad Sci USA 104:17370–17375.ADSCrossRefGoogle Scholar
  56. 56.
    Manley, S., J. M. Gillette, G. H. Patterson, H. Shroff, H. F. Hess, E. Betzig, and J. Lippincott-Schwartz. 2008. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nat Methods 5:155–157.CrossRefGoogle Scholar
  57. 57.
    Jacquier, V., M. Prummer, J. M. Segura, H. Pick, and H. Vogel. 2006. Visualizing odorant receptor trafficking in living cells down to the single-molecule level. Proc Natl Acad Sci USA 103:14325–14330.ADSCrossRefGoogle Scholar
  58. 58.
    James, J. R., S. S. White, R. W. Clarke, A. M. Johansen, P. D. Dunne, D. L. Sleep, W. J. Fitzgerald, S. J. Davis, and D. Klenerman. 2007. Single-molecule level analysis of the subunit composition of the T cell receptor on live T cells. Proc Natl Acad Sci USA 104:17662–17667.ADSCrossRefGoogle Scholar
  59. 59.
    Morimatsu, M., H. Takagi, K. G. Ota, R. Iwamoto, T. Yanagida, and Y. Sako. 2007. Multiple-state reactions between the epidermal growth factor receptor and Grb2 as observed by using single-molecule analysis. Proc Natl Acad Sci USA 104:18013–18018.ADSCrossRefGoogle Scholar
  60. 60.
    Sako, Y., S. Minoghchi, and T. Yanagida. 2000. Single-molecule imaging of EGFR signalling on the surface of living cells. Nat Cell Biol 2:168–172.CrossRefGoogle Scholar
  61. 61.
    Ueda, M., Y. Sako, T. Tanaka, P. Devreotes, and T. Yanagida. 2001. Single-molecule analysis of chemotactic signaling in Dictyostelium cells. Science 294:864–867.ADSCrossRefGoogle Scholar
  62. 62.
    Füreder-Kitzmüller, E., J. Hesse, A. Ebner, H. J. Gruber, and G. J. Schütz. 2005. Non-exponential bleaching of single bioconjugated Cy5 molecules. Chem Phys Lett 404:13–18.ADSCrossRefGoogle Scholar
  63. 63.
    Hecht, E. 1987. Optics. Reading, MA: Addison-Wesley.Google Scholar
  64. 64.
    Enderlein, J., E. Toprak, and P. R. Selvin. 2006. Polarization effect on position accuracy of fluorophore localization. Opt Express 14:8111–8120.ADSCrossRefGoogle Scholar
  65. 65.
    Pohl, D. W., W. Denk, and M. Lanz. 1984. Optical stethoscopy: image recording with resolution λ/20. Appl Phys Lett 44:651–653.ADSCrossRefGoogle Scholar
  66. 66.
    Betzig, E., and J. K. Trautman. 1992. Near-field optics: microscopy, spectroscopy, and surface modification beyond the diffraction limit. Science 257:189–195.ADSCrossRefGoogle Scholar
  67. 67.
    Denk, W., J. H. Strickler, and W. W. Webb. 1990. Two-photon laser scanning fluorescence microscopy. Science 248:73–76.ADSCrossRefGoogle Scholar
  68. 68.
    Klar, T. A., S. Jakobs, M. Dyba, A. Egner, and S. W. Hell. 2000. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc Natl Acad Sci USA 97:8206–8210.ADSCrossRefGoogle Scholar
  69. 69.
    Pawley, J. B., editor. 1995. Handbook of biological confocal microscopy. New York: Plenum Press.Google Scholar
  70. 70.
    Gustafsson, M. G. 2005. Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc Natl Acad Sci USA 102:13081–13086.ADSCrossRefGoogle Scholar
  71. 71.
    Heintzmann, R., T. M. Jovin, and C. Cremer. 2002. Saturated patterned excitation microscopy—a concept for optical resolution improvement. J Opt Soc Am A – Opt Image Sci Vis 19:1599–1609.ADSCrossRefGoogle Scholar
  72. 72.
    Hell, S. W. 2007. Far-field optical nanoscopy. Science 316:1153–1158.ADSCrossRefGoogle Scholar
  73. 73.
    Betzig, E. 1995. Proposed method for molecular optical imaging. Opt Lett 20:237–239.ADSCrossRefGoogle Scholar
  74. 74.
    Bobroff, N. 1986. Position measurement with a resolution and noise-limited instrument. Rev Sci Instrum 57:1152–1157.ADSCrossRefGoogle Scholar
  75. 75.
    Yildiz, A., J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin. 2003. Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300:2061–2065.ADSCrossRefGoogle Scholar
  76. 76.
    Ober, R. J., S. Ram, and E. S. Ward. 2004. Localization accuracy in single-molecule microscopy. Biophys J 86:1185–1200.CrossRefGoogle Scholar
  77. 77.
    Ha, T., T. Enderle, D. S. Chemla, and S. Weiss. 1996. Dual-molecule spectroscopy: molecular rulers for the study of biological macromolecules. IEEE J Sel Top Quant Electr 2: 1115–1128.CrossRefGoogle Scholar
  78. 78.
    van Oijen, A. M., J. Kohler, J. Schmidt, M. Muller, and G. J. Brakenhoff. 1999. Far-field fluorescence microscopy beyond the diffraction limit. J Opt Soc Am A 16:909–915.ADSCrossRefGoogle Scholar
  79. 79.
    Trabesinger, W., G. J. Schütz, H. J. Gruber, H. Schindler, and T. Schmidt. 1999. Detection of individual oligonucleotide pairing by single-molecule microscopy. Anal Chem 71:279–283.CrossRefGoogle Scholar
  80. 80.
    Trabesinger, W., B. Hecht, U. P. Wild, G. J. Schütz, H. Schindler, and T. Schmidt. 2001. Statistical analysis of single-molecule colocalization assays. Anal Chem 73:1100–1105.CrossRefGoogle Scholar
  81. 81.
    Baumgartner, W., G. J. Schütz, J. Wiegand, N. Golenhofen, and D. Drenckhahn. 2003. Cadherin function probed by laser tweezer and single molecule fluorescence in vascular endothelial cells. J Cell Sci 116: 1001–1011.CrossRefGoogle Scholar
  82. 82.
    Yildiz, A., M. Tomishige, R. D. Vale, and P. R. Selvin. 2004. Kinesin walks hand-over-hand. Science 303: 676–678.ADSCrossRefGoogle Scholar
  83. 83.
    Gordon, M. P., T. Ha, and P. R. Selvin. 2004. Single-molecule high-resolution imaging with photobleaching. Proc Natl Acad Sci USA 101:6462–6465.Google Scholar
  84. 84.
    Qu, X., D. Wu, L. Mets, and N. F. Scherer. 2004. Nanometer-localized multiple single-molecule fluorescence microscopy. Proc Natl Acad Sci USA 101:11298–11303.ADSCrossRefGoogle Scholar
  85. 85.
    Patterson, G. H., and J. Lippincott-Schwartz. 2002. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873–1877.ADSCrossRefGoogle Scholar
  86. 86.
    Betzig, E., G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess. 2006. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642–1645.ADSCrossRefGoogle Scholar
  87. 87.
    Hess, S. T., T. P. Girirajan, and M. D. Mason. 2006. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91:4258–4272.CrossRefGoogle Scholar
  88. 88.
    Rust, M., M. Bates, and X. Zhuang. 2006. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3:793–795.CrossRefGoogle Scholar
  89. 89.
    Shroff, H., C. G. Galbraith, J. A. Galbraith, and E. Betzig. 2008. Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics. Nat Methods 5:417–423.CrossRefGoogle Scholar
  90. 90.
    Orrit, M., and J. Bernard. 1990. Single pentacene molecules detected by fluorescence excitation in a p-terphenyl crystal. Phys Rev Lett 65:2716–2719.ADSCrossRefGoogle Scholar
  91. 91.
    Harms, G. S., L. Cognet, P. H. Lommerse, G. A. Blab, and T. Schmidt. 2001. Autofluorescent proteins in single-molecule research: applications to live cell imaging microscopy. Biophys J 80:2396–2408.CrossRefGoogle Scholar
  92. 92.
    Holtzer, L., T. Meckel, and T. Schmidt. 2007. Nanometric three-dimensional tracking of individual quantum dots in cells. Appl Phys Lett 90:053902.Google Scholar
  93. 93.
    Prabhat, P., Z. Gan, J. Chao, S. Ram, C. Vaccaro, S. Gibbons, R. J. Ober, and E. S. Ward. 2007. Elucidation of intracellular recycling pathways leading to exocytosis of the Fc receptor, FcRn, by using multifocal plane microscopy. Proc Natl Acad Sci USA 104:5889–5894.ADSCrossRefGoogle Scholar
  94. 94.
    Ghosh, R. N., and W. W. Webb. 1994. Automated detection and tracking of individual and clustered cell surface low density lipoprotein receptor molecules. Biophys J 66:1301–1318.CrossRefGoogle Scholar
  95. 95.
    Semrau, S., and T. Schmidt. 2006. Particle image correlation spectroscopy (PICS) Retrieving nanometer-scale correlations from high-density single-molecule position data. Biophys J 92:613–621.CrossRefGoogle Scholar
  96. 96.
    Falck, E., T. Rog, M. Karttunen, and I. Vattulainen. 2008. Lateral diffusion in lipid membranes through collective flows. J Am Chem Soc 130:44–45.CrossRefGoogle Scholar
  97. 97.
    Almeida, P. F., W. L. Vaz, and T. E. Thompson. 2005. Lipid diffusion, free area, and molecular dynamics simulations. Biophys J 88:4434–4438.CrossRefGoogle Scholar
  98. 98.
    Falck, E., M. Patra, M. Karttunen, M. T. Hyvonen, and I. Vattulainen. 2005. Response to comment by Almeida et al.: free area theories for lipid bilayers—predictive or not? Biophys J 89:745–752.CrossRefGoogle Scholar
  99. 99.
    Berg, H. C. 1983. Random walks in biology. Princeton, New Jersey: Princeton University Press.Google Scholar
  100. 100.
    Saffman, P. G., and M. Delbruck. 1975. Brownian motion in biological membranes. Proc Natl Acad Sci USA 72:3111–3113.ADSCrossRefGoogle Scholar
  101. 101.
    Hughes, B. D., B. A. Pailthorpe, and L. R. White. 1981. The translational and rotational drag on a cylinder moving in a membrane. J. Fluid. Mech. 110:349–372.MathSciNetADSMATHCrossRefGoogle Scholar
  102. 102.
    Hughes, B. D., B. A. Pailthorpe, L. R. White, and W. H. Sawyer. 1982. Extraction of membrane microviscosity from translational and rotational diffusion coefficients. Biophys J 37:673–676.Google Scholar
  103. 103.
    Petrov, E. P., and P. Schwille. 2008. Translational diffusion in lipid membranes beyond the Saffman-Delbruck approximation. Biophys J 94:L41–43.CrossRefGoogle Scholar
  104. 104.
    Gambin, Y., R. Lopez-Esparza, M. Reffay, E. Sierecki, N. S. Gov, M. Genest, R. S. Hodges, and W. Urbach. 2006. Lateral mobility of proteins in liquid membranes revisited. Proc Natl Acad Sci USA 103: 2098–2102.ADSCrossRefGoogle Scholar
  105. 105.
    Saxton, M. J. 1995. Single-particle tracking: effects of corrals. Biophys J 69:389–398.ADSCrossRefGoogle Scholar
  106. 106.
    Simson, R., E. D. Sheets, and K. Jacobson. 1995. Detection of temporary lateral confinement of membrane proteins using single-particle tracking analysis. Biophys J 69:989–993.CrossRefGoogle Scholar
  107. 107.
    Wieser, S., and G. J. Schütz 2008. Tracking single molecules in the live cell plasma membrane—Do’s and Don’t’s. Methods 46:131–140.Google Scholar
  108. 108.
    Feder, T. J., I. Brust-Mascher, J. P. Slattery, B. Baird, and W. W. Webb. 1996. Constrained diffusion or immobile fraction on cell surfaces: a new interpretation. Biophys J 70:2767–2773.CrossRefGoogle Scholar
  109. 109.
    Smith, P. R., I. E. Morrison, K. M. Wilson, N. Fernandez, and R. J. Cherry. 1999. Anomalous diffusion of major histocompatibility complex class I molecules on HeLa cells determined by single particle tracking. Biophys J 76:3331–3344.CrossRefGoogle Scholar
  110. 110.
    Ritchie, K., X. Y. Shan, J. Kondo, K. Iwasawa, T. Fujiwara, and A. Kusumi. 2005. Detection of non-Brownian diffusion in the cell membrane in single molecule tracking. Biophys J 88:2266–2277.CrossRefGoogle Scholar
  111. 111.
    Guigas, G., and M. Weiss. 2007. Sampling the cell with anomalous diffusion—the discovery of slowness. Biophys J 94:90–94.CrossRefGoogle Scholar
  112. 112.
    Nicolau, D. V., Jr., J. F. Hancock, and K. Burrage. 2007. Sources of anomalous diffusion on cell membranes: a Monte Carlo study. Biophys J 92:1975–1987.CrossRefGoogle Scholar
  113. 113.
    Saxton, M. J. 1996. Anomalous diffusion due to binding: a Monte Carlo study. Biophys J 70:1250–1262.CrossRefGoogle Scholar
  114. 114.
    Saxton, M. J. 2007. A biological interpretation of transient anomalous subdiffusion. II. Reaction kinetics. Biophys J 94:760–771.CrossRefGoogle Scholar
  115. 115.
    Saxton, M. J. 2007. A biological interpretation of transient anomalous subdiffusion. I. Qualitative model. Biophys J 92:1178–1191.CrossRefGoogle Scholar
  116. 116.
    Saxton, M. J. 1993. Lateral diffusion in an archipelago. Single-particle diffusion. Biophys J 64:1766–1780.CrossRefGoogle Scholar
  117. 117.
    Martin, D. S., M. B. Forstner, and J. A. Kas. 2002. Apparent subdiffusion inherent to single particle tracking. Biophys J 83:2109–2117.CrossRefGoogle Scholar
  118. 118.
    Sako, Y., and A. Kusumi. 1995. Barriers for lateral diffusion of transferrin receptor in the plasma membrane as characterized by receptor dragging by laser tweezers: fence versus tether. J Cell Biol 129: 1559–1574.CrossRefGoogle Scholar
  119. 119.
    Daumas, F., N. Destainville, C. Millot, A. Lopez, D. Dean, and L. Salome. 2003. Confined diffusion without fences of a g-protein–coupled receptor as revealed by single particle tracking. Biophys J 84:356–366.CrossRefGoogle Scholar
  120. 120.
    Lillemeier, B. F., J. R. Pfeiffer, Z. Surviladze, B. S. Wilson, and M. M. Davis. 2006. Plasma membrane-associated proteins are clustered into islands attached to the cytoskeleton. Proc Natl Acad Sci USA 103: 18992–18997.ADSCrossRefGoogle Scholar
  121. 121.
    King, M. R. 2004. Apparent 2-D diffusivity in a ruffled cell membrane. J Theor Biol 227:323–326.CrossRefGoogle Scholar
  122. 122.
    Reister, E., and U. Seifert. 2005. Lateral diffusion of a protein on a fluctuating membrane. Europhys Lett 71:859–865.ADSCrossRefGoogle Scholar
  123. 123.
    Aizenbud, B. M., and N. D. Gershon. 1982. Diffusion of molecules on biological membranes of nonplanar form. A theoretical study. Biophys J 38:287–293.CrossRefGoogle Scholar
  124. 124.
    Rustom, A., R. Saffrich, I. Markovic, P. Walther, and H. H. Gerdes. 2004. Nanotubular highways for intercellular organelle transport. Science 303:1007–1010.ADSCrossRefGoogle Scholar
  125. 125.
    Goulian, M., and S. M. Simon. 2000. Tracking single proteins within cells. Biophys J 79:2188–2198.CrossRefGoogle Scholar
  126. 126.
    Destainville, N., and L. Salome. 2006. Quantification and correction of systematic errors due to detector time-averaging in single-molecule tracking experiments. Biophys J 90:L17–19.CrossRefGoogle Scholar
  127. 127.
    Weiss, S. 1999. Fluorescence spectroscopy of single biomolecules. Science 283:1676–1683.ADSCrossRefGoogle Scholar
  128. 128.
    Baumgart, T., S. T. Hess, and W. W. Webb. 2003. Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension. Nature 425:821–824.ADSCrossRefGoogle Scholar
  129. 129.
    Cognet, L., G. S. Harms, G. A. Blab, P. H. M. Lommerse, and T. Schmidt. 2000. Simultaneous dual-color and dual-polarization imaging of single molecules. Appl Phys Lett 77:4052–4054.ADSCrossRefGoogle Scholar
  130. 130.
    Jacobson, K., E. D. Sheets, and R. Simson. 1995. Revisiting the fluid mosaic model of membranes. Science 268:1441–1442.ADSCrossRefGoogle Scholar
  131. 131.
    Hac, A. E., H. M. Seeger, M. Fidorra, and T. Heimburg. 2005. Diffusion in two-component lipid membranes—a fluorescence correlation spectroscopy and Monte Carlo simulation study. Biophys J 88: 317–333.CrossRefGoogle Scholar
  132. 132.
    Loose, M., E. Fischer-Friedrich, J. Ries, K. Kruse, and P. Schwille. 2008. Spatial regulators for bacterial cell division self-organize into surface waves in vitro. Science 320:789–792.ADSCrossRefGoogle Scholar
  133. 133.
    Benson, R. C., R. A. Meyer, M. E. Zaruba, and G. M. McKhann. 1979. Cellular autofluorescence—is it due to flavins? J Histochem Cytochem 27:44–48.CrossRefGoogle Scholar
  134. 134.
    Konig, K., P. T. So, W. W. Mantulin, B. J. Tromberg, and E. Gratton. 1996. Two-photon excited lifetime imaging of autofluorescence in cells during UVA and NIR photostress. J Microsc 183:197–204.Google Scholar
  135. 135.
    Andersson, H., T. Baechi, M. Hoechl, and C. Richter. 1998. Autofluorescence of living cells. J Microsc 191(Pt 1):1–7.Google Scholar
  136. 136.
    Schnell, S. A., W. A. Staines, and M. W. Wessendorf. 1999. Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue. J Histochem Cytochem 47:719–730.CrossRefGoogle Scholar
  137. 137.
    Moertelmaier, M. A., E. J. Kögler, J. Hesse, M. Sonnleitner, L. A. Huber, and G. J. Schütz. 2002. Single molecule microscopy in living cells: subtraction of autofluorescence based on two color recording. Single Mol. 3:225–231.ADSCrossRefGoogle Scholar
  138. 138.
    Seisenberger, G., M. U. Ried, T. Endress, H. Buning, M. Hallek, and C. Brauchle. 2001. Real-time single-molecule imaging of the infection pathway of an adeno-associated virus. Science 294:1929–1932.ADSCrossRefGoogle Scholar
  139. 139.
    van den Berg, C. W., T. Cinek, M. B. Hallett, V. Horejsi, and B. P. Morgan. 1995. Exogenous glycosyl phosphatidylinositol-anchored CD59 associates with kinases in membrane clusters on U937 cells and becomes Ca(2+)-signaling competent. J Cell Biol 131:669–677.CrossRefGoogle Scholar
  140. 140.
    Shaner, N. C., P. A. Steinbach, and R. Y. Tsien. 2005. A guide to choosing fluorescent proteins. Nat Methods 2:905–909.CrossRefGoogle Scholar
  141. 141.
    Gronemeyer, T., G. Godin, and K. Johnsson. 2005. Adding value to fusion proteins through covalent labelling. Curr Opin Biotechnol 16:453–458.CrossRefGoogle Scholar
  142. 142.
    Freudenthaler, G., M. Axmann, H. Schindler, B. Pragl, H. G. Knaus, and G. J. Schütz. 2002. Ultrasensitive pharmacological characterisation of the voltage-gated potassium channel K(V)1.3 studied by single-molecule fluorescence microscopy. Histochem Cell Biol 117:197–202.CrossRefGoogle Scholar
  143. 143.
    Nechyporuk-Zloy, V., P. Dieterich, H. Oberleithner, C. Stock, and A. Schwab. 2008. Dynamics of single potassium channel proteins in the plasma membrane of migrating cells. Am J Physiol Cell Physiol 294: C1096–1102.CrossRefGoogle Scholar
  144. 144.
    Howarth, M., W. Liu, S. Puthenveetil, Y. Zheng, L. F. Marshall, M. M. Schmidt, K. D. Wittrup, M. G. Bawendi, and A. Y. Ting. 2008. Monovalent, reduced-size quantum dots for imaging receptors on living cells. Nat Methods 5:397–399.CrossRefGoogle Scholar
  145. 145.
    Morrisett, J. D., H. J. Pownall, R. T. Plumlee, L. C. Smith, and Z. E. Zehner. 1975. Multiple thermotropic phase transitions in Escherichia coli membranes and membrane lipids. A comparison of results obtained by nitroxyl stearate paramagnetic resonance, pyrene excimer fluorescence, and enzyme activity measurements. J Biol Chem 250:6969–6976.Google Scholar
  146. 146.
    Brown, D. A., and J. K. Rose. 1992. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell 68:533–544.CrossRefGoogle Scholar
  147. 147.
    Melkonian, K. A., T. Chu, L. B. Tortorella, and D. A. Brown. 1995. Characterization of proteins in detergent-resistant membrane complexes from Madin-Darby canine kidney epithelial cells. Biochemistry 34: 16161–16170.CrossRefGoogle Scholar
  148. 148.
    Lisanti, M. P., P. E. Scherer, J. Vidugiriene, Z. Tang, A. Hermanowski-Vosatka, Y. H. Tu, R. F. Cook, and M. Sargiacomo. 1994. Characterization of caveolin-rich membrane domains isolated from an endothelial-rich source: implications for human disease. J Cell Biol 126:111–126.CrossRefGoogle Scholar
  149. 149.
    Horejsi, V., K. Drbal, M. Cebecauer, J. Cerny, T. Brdicka, P. Angelisova, and H. Stockinger. 1999. GPI-microdomains: a role in signalling via immunoreceptors. Immunol Today 20:356–361.CrossRefGoogle Scholar
  150. 150.
    Brown, R. E. 1998. Sphingolipid organization in biomembranes: what physical studies of model membranes reveal. J Cell Sci 111(Pt 1):1–9.Google Scholar
  151. 151.
    Schroeder, R., E. London, and D. Brown. 1994. Interactions between saturated acyl chains confer detergent resistance on lipids and glycosylphosphatidylinositol (GPI)-anchored proteins: GPI-anchored proteins in liposomes and cells show similar behavior. Proc Natl Acad Sci USA 91:12130–12134.ADSCrossRefGoogle Scholar
  152. 152.
    Brown, D. A., and E. London. 1998. Structure and origin of ordered lipid domains in biological membranes. J Membr Biol 164:103–114.CrossRefGoogle Scholar
  153. 153.
    Brown, D. A., and E. London. 1998. Functions of lipid rafts in biological membranes. Annu Rev Cell Dev Biol 14:111–136.CrossRefGoogle Scholar
  154. 154.
    Resh, M. D. 1999. Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim Biophys Acta 1451:1–16.CrossRefGoogle Scholar
  155. 155.
    Peirce, M., and H. Metzger. 2000. Detergent-resistant microdomains offer no refuge for proteins phosphorylated by the IgE receptor. J Biol Chem 275:34976–34982.CrossRefGoogle Scholar
  156. 156.
    Kurzchalia, T. V., E. Hartmann, and P. Dupree. 1995. Guilt by insolubility—does a protein’s detergent insolubility reflect a caveolar location? Trends Cell Biol 5:187–189.CrossRefGoogle Scholar
  157. 157.
    Heerklotz, H. 2002. Triton promotes domain formation in lipid raft mixtures. Biophys J 83:2693–2701.CrossRefGoogle Scholar
  158. 158.
    London, E., and D. A. Brown. 2000. Insolubility of lipids in triton X-100: physical origin and relationship to sphingolipid/cholesterol membrane domains (rafts). Biochim Biophys Acta 1508:182–195.CrossRefGoogle Scholar
  159. 159.
    Li, X. M., J. M. Smaby, M. M. Momsen, H. L. Brockman, and R. E. Brown. 2000. Sphingomyelin interfacial behavior: the impact of changing acyl chain composition. Biophys J 78:1921–1931.CrossRefGoogle Scholar
  160. 160.
    Edidin, M., S. C. Kuo, and M. P. Sheetz. 1991. Lateral movements of membrane glycoproteins restricted by dynamic cytoplasmic barriers. Science 254:1379–1382.ADSCrossRefGoogle Scholar
  161. 161.
    Lee, G. M., F. Zhang, A. Ishihara, C. L. McNeil, and K. A. Jacobson. 1993. Unconfined lateral diffusion and an estimate of pericellular matrix viscosity revealed by measuring the mobility of gold-tagged lipids. J Cell Biol 120:25–35.CrossRefGoogle Scholar
  162. 162.
    Tomishige, M., Y. Sako, and A. Kusumi. 1998. Regulation mechanism of the lateral diffusion of band 3 in erythrocyte membranes by the membrane skeleton. J Cell Biol 142:989–1000.CrossRefGoogle Scholar
  163. 163.
    Simson, R., B. Yang, S. E. Moore, P. Doherty, F. S. Walsh, and K. A. Jacobson. 1998. Structural mosaicism on the submicron scale in the plasma membrane. Biophys J 74:297–308.CrossRefGoogle Scholar
  164. 164.
    Sako, Y., and A. Kusumi. 1994. Compartmentalized structure of the plasma membrane for receptor movements as revealed by a nanometer-level motion analysis. J Cell Biol 125:1251–1264.CrossRefGoogle Scholar
  165. 165.
    Suzuki, K. G., T. K. Fujiwara, M. Edidin, and A. Kusumi. 2007. Dynamic recruitment of phospholipase C{gamma} at transiently immobilized GPI-anchored receptor clusters induces IP3-Ca2+ signaling: single-molecule tracking study 2. J Cell Biol 177:731–742.CrossRefGoogle Scholar
  166. 166.
    Pike, L. J. 2006. Rafts defined. J Lipid Res 47:1597–1598.Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Thomas Schmidt
    • 1
  • Gerhard J. Schütz
    • 2
  1. 1.Physics of Life Processes, Leiden Institute of Physics, Leiden University, Niels Bohrweg 22333 CA LeidenThe Netherlands
  2. 2.Biophysics Institute, Johannes Kepler University Linz, Altenbergerstr. 69A-4040 LinzAustria

Personalised recommendations