Advertisement

Scanning Fluorescence Correlation Spectroscopy on Biomembranes

  • Eduard Hermann
  • Jonas Ries
  • Ana J. García-Sáez
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1232)

Abstract

Fluorescence correlation spectroscopy (FCS) is a powerful quantitative method to study dynamical properties of biophysical systems. It exploits the temporal autocorrelation of fluorescence intensity fluctuations originating from a tiny volume (~fL). A theoretical model function can be then fitted to the measured auto-correlation curve to obtain physical parameters such as local concentration and diffusion time. However, the application of FCS on membranes is coupled to several difficulties like accurate positioning and stability of the set-up.

In this book chapter, we explain the theoretical framework of point FCS and Scanning FCS (SFCS), which is a variation especially suitable for membrane studies. We present a list of materials necessary for SFCS studies on Giant Unilamellar Vesicles (GUVs). Finally, we provide simple protocols for the preparation of GUVs, calibration of the microscope setup, and acquisition and analysis of SFCS data to determine diffusion coefficients and concentrations of fluorescent particles embedded in lipid membranes.

Key words

Fluorescence correlation spectroscopy Giant unilamellar vesicles Scanning FCS Diffusion coefficient Membrane 

References

  1. 1.
    Rigler R, Elson E (2001) Fluorescence correlation spectroscopy: theory and applications. Springer, New YorkCrossRefGoogle Scholar
  2. 2.
    Bacia K, Schwille P (2003) A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy. Methods 29:74–85PubMedCrossRefGoogle Scholar
  3. 3.
    Magde D, Elson EL, Webb WW (1972) Thermodynamic fluctuations in a reacting system—measurement by fluorescence correlation spectroscopy. Phys Rev Lett 29:705–708CrossRefGoogle Scholar
  4. 4.
    Aragon SR, Pecora R (1975) Fluorescence correlation spectroscopy and Brownian rotational diffusion. Biopolymers 14:119–137CrossRefGoogle Scholar
  5. 5.
    Elson EL, Magde D (1974) Fluorescence correlation spectroscopy. I. Conceptual basis and theory. Biopolymers 13:1–27CrossRefGoogle Scholar
  6. 6.
    Koppel D (1974) Statistical accuracy in fluorescence correlation spectroscopy. Phys Rev A 10:1938–1945CrossRefGoogle Scholar
  7. 7.
    Koppel DE, Axelrod D, Schlessinger J et al (1976) Dynamics of fluorescence marker concentration as a probe of mobility. Biophys J 16:1315–1329PubMedPubMedCentralCrossRefGoogle Scholar
  8. 8.
    Magde D, Elson EL, Webb WW (1974) Fluorescence correlation spectroscopy. II. An experimental realization. Biopolymers 13:29–61PubMedCrossRefGoogle Scholar
  9. 9.
    Magde D, Elson EL, Webb WW (1978) Fluorescence correlation spectroscopy. III. Uniform translation and lamellar flow. Biopolymers 17:361–376CrossRefGoogle Scholar
  10. 10.
    Fahey PF, Koppel DE, Barak LS et al (1977) Lateral diffusion in planar lipid bilayers. Science 195:305–306PubMedCrossRefGoogle Scholar
  11. 11.
    Schwille P, Korlach J, Webb WW (1999) Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes. Cytometry 36:176–182PubMedCrossRefGoogle Scholar
  12. 12.
    Korlach J, Schwille P, Webb WW et al (1999) Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy. Proc Natl Acad Sci 96:8461–8466PubMedPubMedCentralCrossRefGoogle Scholar
  13. 13.
    Bacia K, Kim SA, Schwille P (2006) Fluorescence cross-correlation spectroscopy in living cells. Nat Methods 3:83–89PubMedCrossRefGoogle Scholar
  14. 14.
    Haustein E, Schwille P (2003) Ultrasensitive investigations of biological systems by fluorescence correlation spectroscopy. Methods 29:153–166PubMedCrossRefGoogle Scholar
  15. 15.
    Hess ST, Huang S, Heikal AA et al (2002) Biological and chemical applications of fluorescence correlation spectroscopy: a review. Biochemistry 41:697–705PubMedCrossRefGoogle Scholar
  16. 16.
    Kim SA, Heinze KG, Schwille P (2007) Fluorescence correlation spectroscopy in living cells. Nat Methods 4:963–973PubMedCrossRefGoogle Scholar
  17. 17.
    Petrov E, Schwille P (2008) State of the art and novel trends in fluorescence correlation spectroscopy. In: Resch-Genger U (ed) Standardization and quality assurance in fluorescence measurements II: Bioanalytical and biomedical applications. Springer, BerlinGoogle Scholar
  18. 18.
    Ries J, Schwille P (2008) New concepts for fluorescence correlation spectroscopy on membranes. Phys Chem Chem Phys 10:3487–3497PubMedCrossRefGoogle Scholar
  19. 19.
    Schwille P (2001) Fluorescence correlation spectroscopy and its potential for intracellular applications. Cell Biochem Biophys 34:383–408PubMedCrossRefGoogle Scholar
  20. 20.
    Thompson NL, Lieto AM, Allen NW (2002) Recent advances in fluorescence correlation spectroscopy. Curr Opin Struct Biol 12:634–641PubMedCrossRefGoogle Scholar
  21. 21.
    Rigler R, Mets Ü, Widengren J (1993) Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion. Eur Biophys J 22:169–175Google Scholar
  22. 22.
    Ries J, Weidemann T, Schwille P (2012) Fluorescence correlation spectroscopy. In: Egelman E (ed) Comprehensive biophysics. Academic, New York, pp 210–245CrossRefGoogle Scholar
  23. 23.
    Tcherniak A, Reznik C, Link S et al (2009) Fluorescence correlation spectroscopy: criteria for analysis in complex systems. Anal Chem 81:746–754PubMedCrossRefGoogle Scholar
  24. 24.
    Ries J, Chiantia S, Schwille P (2009) Accurate determination of membrane dynamics with line-scan FCS. Biophys J 96:1999–2008PubMedPubMedCentralCrossRefGoogle Scholar
  25. 25.
    Ries J, Schwille P (2006) Studying slow membrane dynamics with continuous wave scanning fluorescence correlation spectroscopy. Biophys J 91:1915–1924PubMedPubMedCentralCrossRefGoogle Scholar
  26. 26.
    Bacia K, Schwille P (2007) Practical guidelines for dual-color fluorescence cross-correlation spectroscopy. Nat Protoc 2:2842–2856PubMedCrossRefGoogle Scholar
  27. 27.
    Dertinger T, Loman A, Ewers B et al (2008) The optics and performance of dual-focus fluorescence correlation spectroscopy. Opt Express 16:14353–14368PubMedCrossRefGoogle Scholar
  28. 28.
    Ries J, Petrásek Z, García-Sáez AJ et al (2010) A comprehensive framework for fluorescence cross-correlation spectroscopy. N J Phys 12Google Scholar
  29. 29.
    Angelova MI, Dimitrov DS (1986) Liposome electroformation. Faraday Discuss 81:303CrossRefGoogle Scholar
  30. 30.
    Chiantia S, Schwille P, Klymchenko AS et al (2011) Asymmetric GUVs prepared by MbetaCD-mediated lipid exchange: an FCS study. Biophys J 100:L1–L3PubMedPubMedCentralCrossRefGoogle Scholar
  31. 31.
    Chiantia S, Ries J, Schwille P (2009) Fluorescence correlation spectroscopy in membrane structure elucidation. Biochim Biophys Acta 1788:225–233PubMedCrossRefGoogle Scholar
  32. 32.
    Bacia K, Schuette CG, Kahya N et al (2004) SNAREs prefer liquid-disordered over “raft” (liquid-ordered) domains when reconstituted into giant unilamellar vesicles. J Biol Chem 279:37951–37955PubMedCrossRefGoogle Scholar
  33. 33.
    Doeven MK, Folgering JH, Krasnikov V et al (2005) Distribution, lateral mobility and function of membrane proteins incorporated into giant unilamellar vesicles. Biophys J 88:1134–1142PubMedPubMedCentralCrossRefGoogle Scholar
  34. 34.
    Garcia-Saez AJ, Ries J, Orzaez M et al (2009) Membrane promotes tBID interaction with BCL(XL). Nat Struct Mol Biol 16:1178–1185PubMedCrossRefGoogle Scholar
  35. 35.
    Kahya N, Wiersma DA, Poolman B et al (2002) Spatial organization of bacteriorhodopsin in model membranes. Light-induced mobility changes. J Biol Chem 277:39304–39311PubMedCrossRefGoogle Scholar
  36. 36.
    Steringer JP, Bleicken S, Andreas H et al (2012) Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-dependent oligomerization of fibroblast growth factor 2 (FGF2) triggers the formation of a lipidic membrane pore implicated in unconventional secretion. J Biol Chem 287:27659–27669PubMedPubMedCentralCrossRefGoogle Scholar
  37. 37.
    Montes LR, Alonso A, Goni FM et al (2007) Giant unilamellar vesicles electroformed from native membranes and organic lipid mixtures under physiological conditions. Biophys J 93:3548–3554PubMedPubMedCentralCrossRefGoogle Scholar
  38. 38.
    Magatti D, Ferri F (2001) Fast multi-t real-time software correlator for dynamic light scattering. Appl Opt 40:4011–4021PubMedCrossRefGoogle Scholar
  39. 39.
    Chiantia S, Kahya N, Schwille P (2007) Raft domain reorganization driven by short- and long-chain ceramide: a combined AFM and FCS study. Langmuir 23:7659–7665PubMedCrossRefGoogle Scholar
  40. 40.
    Marks KM, Nolan GP (2006) Chemical labeling strategies for cell biology. Nat Methods 3:591–596PubMedCrossRefGoogle Scholar
  41. 41.
    Prescher JA, Bertozzi CR (2005) Chemistry in living systems. Nat Chem Biol 1:13–21PubMedCrossRefGoogle Scholar
  42. 42.
    Angelova MI, Dimitrov DS (1987) Swelling of charged lipids and formation of liposomes on electrode surfaces. Mol Cryst Liq Cryst 152:89–104Google Scholar
  43. 43.
    Dimitrov DS, Angelova MI (1988) Lipid swelling and liposome formation mediated by electric fields. Bioelectrochem Bioenerg 19:323–336CrossRefGoogle Scholar
  44. 44.
    Angelova MI, Soléau S, Méléard P (1992) Preparation of giant vesicles by external AC fields. Kinetics and applications. Prog Coll Polym Sci 89:127–131CrossRefGoogle Scholar
  45. 45.
    Politano TJ, Froude VE, Jing B et al (2010) AC-electric field dependent electroformation of giant lipid vesicles. Colloids Surf B Biointerfaces 79:75–82PubMedCrossRefGoogle Scholar
  46. 46.
    Enderlein J, Gregor I, Patra D et al (2005) Performance of fluorescence correlation spectroscopy for measuring diffusion and concentration. Chemphyschem 6:2324–2336PubMedCrossRefGoogle Scholar
  47. 47.
    Petrasek Z, Schwille P (2008) Precise measurement of diffusion coefficients using scanning fluorescence correlation spectroscopy. Biophys J 94:1437–1448PubMedPubMedCentralCrossRefGoogle Scholar
  48. 48.
    Culbertson CT, Jacobson SC, Michael Ramsey J (2002) Diffusion coefficient measurements in microfluidic devices. Talanta 56:365–373PubMedCrossRefGoogle Scholar
  49. 49.
    Dertinger T, Pacheco V, von der Hocht I et al (2007) Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements. Chemphyschem 8:433–443PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Eduard Hermann
    • 1
    • 2
  • Jonas Ries
    • 3
  • Ana J. García-Sáez
    • 1
    • 2
  1. 1.Max Planck Institute for Intelligent SystemsStuttgartGermany
  2. 2.German Cancer Research Center, BioQuantHeidelbergGermany
  3. 3.European Molecular Biology LaboratoryHeidelbergGermany

Personalised recommendations