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Fluorescence cross-correlation spectroscopy in living cells

Nature Methods volume 3, pages 83–89 (2006)Cite this article

Abstract

Cell biologists strive to characterize molecular interactions directly in the intracellular environment. The intrinsic resolution of optical microscopy, however, allows visualization of only coarse subcellular localization. By extracting information from molecular dynamics, fluorescence cross-correlation spectroscopy (FCCS) grants access to processes on a molecular scale, such as diffusion, binding, enzymatic reactions and codiffusion, and has become a valuable tool for studies in living cells. Here we review basic principles of FCCS and focus on seminal applications, including examples of intracellular signaling and trafficking. We consider FCCS in the context of fluorescence resonance energy transfer and multicolor imaging techniques and discuss application strategies and recent technical advances.

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Figure 1: Parameters assessed by FCS and FCCS.
Figure 2: The principle of FCS and FCCS measurements.

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References

  1. Rigler, R. & Elson, E.L. (eds.) Fluorescence Correlation Spectroscopy: Theory and Applications (Springer, Berlin, 2001).

    Book  Google Scholar 

  2. Thompson, N.L. Fluorescence correlation spectroscopy. In Topics in Fluorescence Spectroscopy, Volume 1: Techniques. (Lakowicz, J.R. ed.) 337–378 (Plenum, New York, 1991).

    Google Scholar 

  3. Haustein, E. & Schwille, P. Single-molecule spectroscopic methods. Curr. Opin. Struct. Biol. 14, 531–540 (2004).

    Article  CAS  Google Scholar 

  4. Bacia, K. & Schwille, P. A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy. Methods 29, 74–85 (2003).

    Article  CAS  Google Scholar 

  5. Kim, S.A. & Schwille, P. Intracellular applications of fluorescence correlation spectroscopy: prospects for neuroscience. Curr. Opin. Neurobiol. 13, 583–590 (2003).

    Article  CAS  Google Scholar 

  6. Krichevsky, O. & Bonnet, G. Fluorescence correlation spectroscopy: the technique and its applications. Rep. Prog. Phys. 65, 251–297 (2002).

    Article  CAS  Google Scholar 

  7. Schwille, P. Fluorescence correlation spectroscopy and its potential for intracellular applications. Cell Biochem. Biophys. 34, 383–408 (2001).

    Article  CAS  Google Scholar 

  8. Bacia, K., Scherfeld, D., Kahya, N. & Schwille, P. Fluorescence correlation spectroscopy relates rafts in model and native membranes. Biophys. J. 87, 1034–1043 (2004).

    Article  CAS  Google Scholar 

  9. Dauty, E. & Verkman, A.S. Actin cytoskeleton as the principal determinant of size-dependent DNA mobility in cytoplasm. J. Biol. Chem. 280, 7823–7828 (2005).

    Article  CAS  Google Scholar 

  10. Rusu, L., Gambhir, A., McLaughlin, S. & Radler, J. Fluorescence correlation spectroscopy studies of peptide and protein binding to phospholipid vesicles. Biophys. J. 87, 1044–1053 (2004).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  12. Bacia, K., Majoul, I.V. & Schwille, P. Probing the endocytic pathway in live cells using dual-color fluorescence cross-correlation analysis. Biophys. J. 83, 1184–1193 (2002).

    Article  CAS  Google Scholar 

  13. Rigler, R. et al. Specific binding of proinsulin C-peptide to human cell membranes. Proc. Natl. Acad. Sci. USA 96, 13318–13323 (1999).

    Article  CAS  Google Scholar 

  14. Briddon, S.J. et al. Quantitative analysis of the formation and diffusion of A1-adenosine receptor-antagonist complexes in single living cells. Proc. Natl. Acad. Sci. USA 101, 4673–4678 (2004).

    Article  CAS  Google Scholar 

  15. Schwille, P., Meyer-Almes, F.J. & Rigler, R. Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution. Biophys. J. 72, 1878–1886 (1997).

    Article  CAS  Google Scholar 

  16. Kettling, U., Koltermann, A., Schwille, P. & Eigen, M. Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy. Proc. Natl. Acad. Sci. USA 95, 1416–1420 (1998).

    Article  CAS  Google Scholar 

  17. Kohl, T., Heinze, K.G., Kuhlemann, R., Koltermann, A. & Schwille, P. A protease assay for two-photon crosscorrelation and FRET analysis based solely on fluorescent proteins. Proc. Natl. Acad. Sci. USA 99, 12161–12166 (2002).

    Article  CAS  Google Scholar 

  18. Saito, K., Wada, I., Tamura, M. & Kinjo, M. Direct detection of caspase-3 activation in single live cells by cross-correlation analysis. Biochem. Biophys. Res. Commun. 324, 849–854 (2004).

    Article  CAS  Google Scholar 

  19. Kohl, T., Haustein, E. & Schwille, P. Determining protease activity in vivo by fluorescencecross-correlation analysis. Biophys. J. 89, 2770–2782 (2005).

    Article  CAS  Google Scholar 

  20. Kim, S.A., Heinze, K.G., Waxham, M.N. & Schwille, P. Intracellular calmodulin availability accessed with two-photon cross-correlation. Proc. Natl. Acad. Sci. USA 101, 105–110 (2004).

    Article  CAS  Google Scholar 

  21. Kim, S.A., Heinze, K.G., Bacia, K., Waxham, M.N. & Schwille, P. Two-photon cross-correlation analysis of intracellular reactions with variable stoichiometry. Biophys. J. 88, 4319–4336 (2005).

    Article  CAS  Google Scholar 

  22. Baudendistel, N., Muller, G., Waldeck, W., Angel, P. & Langowski, J. Two-hybrid fluorescence cross-correlation spectroscopy detects protein-protein interactions in vivo. ChemPhysChem 6, 984–990 (2005).

    Article  CAS  Google Scholar 

  23. Kirchhausen, T. Three ways to make a vesicle. Nat. Rev. Mol. Cell Biol. 1, 187–198 (2000).

    Article  CAS  Google Scholar 

  24. Amediek, A., Haustein, E., Scherfeld, D. & Schwille, P. Scanning dual-color cross-correlation analysis for dynamic co-localization studies of immobile molecules. Single Molecules 3, 201–210 (2002).

    Article  CAS  Google Scholar 

  25. Ruan, Q., Cheng, M.A., Levi, M., Gratton, E. & Mantulin, W.W. Spatial-temporal studies of membrane dynamics: scanning fluorescence correlation spectroscopy (SFCS). Biophys. J. 87, 1260–1267 (2004).

    Article  CAS  Google Scholar 

  26. Hebert, B., Costantino, S. & Wiseman, P.W. Spatiotemporal image correlation spectroscopy (STICS) theory, verification, and application to protein velocity mapping in living CHO cells. Biophys. J. 88, 3601–3614 (2005).

    Article  CAS  Google Scholar 

  27. Digman, M.A. et al. Measuring fast dynamics in solutions and cells with a laser scanning microscope. Biophys. J. 89, 1317–1327 (2005).

    Article  CAS  Google Scholar 

  28. Bastiaens, P.I.H. & Squire, A. Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell. Trends Cell Biol. 9, 48–52 (1999).

    Article  CAS  Google Scholar 

  29. Jares-Erijman, E.A. & Jovin, T.M. FRET imaging. Nat. Biotechnol. 21, 1387–1395 (2003).

    Article  CAS  Google Scholar 

  30. Zal, T. & Gascoigne, N.R. Photobleaching-corrected FRET efficiency imaging of live cells. Biophys. J. 86, 3923–3939 (2004).

    Article  CAS  Google Scholar 

  31. Weisshart, K., Jungel, V. & Briddon, S.J. The LSM 510 Meta-ConfoCor2 system: An integrated imaging and spectroscopic platform for single-molecule detection. Curr. Pharm. Biotechnol. 5, 135–154 (2004).

    Article  CAS  Google Scholar 

  32. Bacia, K., Schuette, C.G., Kahya, N., Jahn, R. & Schwille, P. SNAREs prefer liquid-disordered over 'raft' (liquid-ordered) domains when reconstituted into giant unilamellar vesicles. J. Biol. Chem. 279, 37951–37955 (2004).

    Article  CAS  Google Scholar 

  33. Jahnz, M., Medina, M.A. & Schwille, P. A novel homogenous assay for topoisomerase II action and inhibition. ChemBioChem 6, 920–926 (2005).

    Article  CAS  Google Scholar 

  34. Stoevesandt, O., Kohler, K., Fischer, R., Johnston, I.C. & Brock, R. One-step analysis of protein complexes in microliters of cell lysate. Nat. Methods 2, 833–835 (2005).

    Article  CAS  Google Scholar 

  35. Muller, B.K., Zaychikov, E., Brauchle, C. & Lamb, D.C. Pulsed interleaved excitation. Biophys. J. 89, 3508–3522 (2005).

    Article  Google Scholar 

  36. Thews, E. et al. Cross talk free fluorescence cross correlation spectroscopy in live cells. Biophys. J. 89, 2069–2076 (2005).

    Article  CAS  Google Scholar 

  37. Levene, M.J. et al. Zero-mode waveguides for single-molecule analysis at high concentrations. Science 299, 682–686 (2003).

    Article  CAS  Google Scholar 

  38. Hwang, L.C. & Wohland, T. Dual-color fluorescence cross-correlation spectroscopy using single laser wavelength excitation. ChemPhysChem 5, 549–551 (2004).

    Article  CAS  Google Scholar 

  39. Burkhardt, M., Heinze, K.G. & Schwille, P. Four-color fluorescence correlation spectroscopy realized in a grating based detection platform. Opt. Lett. 30, 2226–2268 (2005).

    Article  Google Scholar 

  40. Heinze, K.G., Koltermann, A. & Schwille, P. Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis. Proc. Natl. Acad. Sci. USA 97, 10377–10382 (2000).

    Article  CAS  Google Scholar 

  41. Heinze, K.G., Rarbach, M., Jahnz, M. & Schwille, P. Two-photon fluorescence coincidence analysis: Rapid measurements of enzyme kinetics. Biophys. J. 83, 1671–1681 (2002).

    Article  CAS  Google Scholar 

  42. Heinze, K.G., Jahnz, M. & Schwille, P. Triple-color coincidence analysis: One step further in following higher order molecular complex formation. Biophys. J. 86, 506–516 (2004).

    Article  CAS  Google Scholar 

  43. Chen, Y. et al. Dual-color photon-counting histogram. Biophys. J. 88, 2177–2192 (2005).

    Article  CAS  Google Scholar 

  44. Chen, Y., Wei, L.N. & Muller, J.D. Unraveling protein-protein interactions in living cells with fluorescence fluctuation brightness analysis. Biophys. J. 88, 4366–4377 (2005).

    Article  CAS  Google Scholar 

  45. Saffman, P.G. & Delbruck, M. Brownian motion in biological membranes. Proc. Natl. Acad. Sci. USA 72, 3111–3113 (1975).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank E. Haustein for comments on the manuscript. This work was supported by Europäische Fonds für regionale Entwicklung (4212/04-01) and Human Frontiers (RG P66/20021).

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Authors and Affiliations

  1. Institute of Biophysics, Dresden University of Technology, Tatzberg 47-51, Dresden, D-01307, Germany

    Kirsten Bacia, Sally A Kim & Petra Schwille

  2. Department of Applied and Engineering Physics, Cornell University, Ithaca, 14853, New York, USA

    Sally A Kim

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  1. Kirsten Bacia
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  2. Sally A Kim
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Correspondence to Petra Schwille.

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Bacia, K., Kim, S. & Schwille, P. Fluorescence cross-correlation spectroscopy in living cells. Nat Methods 3, 83–89 (2006). https://doi.org/10.1038/nmeth822

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