Advertisement

Fluorescence Correlation Spectroscopy: Principles and Developments

  • Sergey Ivanchenko
  • Don C. LambEmail author
Conference paper

Abstract

Twenty years ago, fluorescence measurements at low concentrations were difficult due to the weak fluorescence signal and intrinsic fluctuations of the sample. With the development of FCS and its implementation on a confocal microscope, it is possible to use the inherent fluctuations to gain information over the concentration, molecular brightness, microscopic rate constants for reactions and mobility of the measured sample. In recent years, there has been a strong increase in the development and application of fluctuation methods. With pulsed interleaved excitation, stoichiometry information can be obtained and spectral cross-talk can be eliminated from FCCS experiments. An elegant implementation of two-focus FCS has also been introduced to allow absolute measurements of diffusion coefficient without precise knowledge of the psf of the microscope and is less sensitive to the laser excitation intensity and saturation effects. Scanning methods such as Scanning FCS and RICS increase the effective volume, which is advantageous for live-cell measurements where diffusion is slow and photobleaching is a problem. In this article, describe the basics of FCS and its limitations as well as a short discussion of a handful of emerging techniques. There are still many other equally interesting applications of fluorescence fluctuation spectroscopy that we have not been able to touch upon. And, if the past is any indication of the future, there will be a number of novel fluorescence fluctuation spectroscopy methods emerging in the near future.

Keywords

Fluorescence correlation spectroscopy (FCS) ACF ALEX ccRISC FRET PIE 

Abbreviations

2fFCS

Two-focus fluorescence correlation spectroscopy

ACF

Autocorrelation function

ALEX

Alternating laser excitation

CCF

Cross-correlation function

ccRISC

Cross-correlation raster image correlation spectroscopy

FCS

Fluorescence correlation spectroscopy

FCCS

Fluorescence cross-correlation spectroscopy

FRET

Förster resonance energy transfer

PIE

Pulsed interleaved excitation

RISC

Raster image correlation spectroscopy

References

  1. Aragón, S.F. and Pecora, R. (1976) Fluorescence correlation spectroscopy as a probe of molecular dynamics. J. Chem. Phys. 64: 1791–8103.CrossRefGoogle Scholar
  2. Berland, K.M., So, P.T., Chen, Y., Mantulin, W.W. and Gratton, E. (1996) Scanning two-photon fluctuation correlation spectroscopy: particle counting measurements for detection of molecular aggregation. Biophys. J. 71: 410–420.PubMedCrossRefGoogle Scholar
  3. Bismuto, E., Gratton, E. and Lamb, D.C. (2001) Dynamics of ANS binding to tuna apomyoglobin measured with fluorescence correlation spectroscopy. Biophys. J. 81: 3510–3521.PubMedCrossRefGoogle Scholar
  4. Bonnet, G., Krichevsky, O. and Libchaber, A. (1998) Kinetics of conformational fluctuations in DNA hairpin-loops. Proc. Natl. Acad. Sci. USA 95: 8602–8606.PubMedCrossRefGoogle Scholar
  5. Boukobza, E., Sonnenfeld, A. and Haran, G. (2001) Immobilization in surface-tethered lipid vesicles as a new tool for single biomolecule spectroscopy. J. Phys. Chem. B 105: 12165–12170.CrossRefGoogle Scholar
  6. Brinkmeier, M., Dorre, K., Riebeseel, K. and Rigler, R. (1997) Confocal spectroscopy in microstructures. Biophys. Chem. 66: 229–239.PubMedCrossRefGoogle Scholar
  7. Brinkmeier, M., Dorre, K., Stephan, J. and Eigen, M. (1999) Two-beam cross-correlation: a method to characterize transport phenomena in micrometer-sized structures. Anal. Chem. 71: 609–616.CrossRefGoogle Scholar
  8. Brown, C.M., Dalal, R.B., Hebert, B., Digman, M.A., Horwitz, A.R. and Gratton, E. (2008) Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope. J. Microsc. 229: 78–91.PubMedCrossRefGoogle Scholar
  9. Brown, R.H. and Twiss, R.Q. (1956) Correlation between photons in two coherent beams of light. Nature 177: 27–29.CrossRefGoogle Scholar
  10. Dertinger, T., Pacheco, V., von der Hocht, I., Hartmann, R., Gregor, I. and Enderlein, J. (2007) Two-focus fluorescence correlation spectroscopy: a new tool for accurate and absolute diffusion measurements. Chemphyschem 8: 433–443.PubMedCrossRefGoogle Scholar
  11. Dickson, R.M., Cubitt, A.B., Tsien, R.Y. and Moerner, W.E. (1997) On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature 388: 355–358.PubMedCrossRefGoogle Scholar
  12. Digman, M.A., Brown, C.M., Sengupta, P., Wiseman, P.W., Horwitz, A.R. and Gratton, E. (2005a). Measuring fast dynamics in solutions and cells with a laser scanning microscope. Biophys. J. 89: 1317–1327.PubMedCrossRefGoogle Scholar
  13. Digman, M.A., Sengupta, P., Wiseman, P.W., Brown, C.M., Horwitz, A.R. and Gratton, E. (2005b). Fluctuation correlation spectroscopy with a laser-scanning microscope: exploiting the hidden time structure. Biophys. J. 88: L33–36.CrossRefGoogle Scholar
  14. Digman, M.A., Dalal, R., Horwitz, A.F. and Gratton, E. (2008) Mapping the number of molecules and brightness in the laser scanning microscope. Biophys. J. 94: 2320–2332.PubMedCrossRefGoogle Scholar
  15. Digman, M.A., Wiseman, P.W., Choi, C., Horwitz, A.R. and Gratton, E. (2009a). Stoichiometry of molecular complexes at adhesions in living cells. Proc. Natl. Acad. Sci. USA 106: 2170–2175.PubMedCrossRefGoogle Scholar
  16. Digman, M.A., Wiseman, P.W., Horwitz, A.R. and Gratton, E. (2009b). Detecting protein complexes in living cells from laser scanning confocal image sequences by the cross correlation raster image spectroscopy method. Biophys. J. 96: 707–716.PubMedCrossRefGoogle Scholar
  17. Ehrenberg, M. and Rigler, R. (1974) Rotational brownian motion and fluorescence intensity fluctuations. Chem. Phys. 4: 390–401.CrossRefGoogle Scholar
  18. Ehrenberg, M. and Rigler, R. (1976) Fluorescence correlation spectroscopy applied to rotational diffusion of macromolecules. Q. Rev. Biophys. 9: 69–81.PubMedCrossRefGoogle Scholar
  19. Eigen, M. and Rigler, R. (1994) Sorting single molecules: application to diagnostics and evolutionary biotechnology. Proc. Natl. Acad. Sci. USA 91: 5740–5747.PubMedCrossRefGoogle Scholar
  20. Einstein, A. (1905) Über die von der molekularkinetischen Theorie der Wärme geforderte Bewegung von in ruhenden Flüssigkeiten suspendierten Teilchen. Ann. Phys. 17: 549–560.CrossRefGoogle Scholar
  21. Elson, E.L. and Magde, D. (1974) Fluorescence correlation spectroscopy. I. Conceptual basis and theory. Biopolymers 13: 1–27.CrossRefGoogle Scholar
  22. Felekyan, S., Kuhnemuth, R., Kudryavtsev, V., Sandhagen, C., Becker, W. and Seidel, C.A.M. (2005) Full correlation from picoseconds to seconds by time-resolved and time-correlated single photon detection. Rev. Sci. Instrum. 76: 083104–083114.CrossRefGoogle Scholar
  23. Ha, T., Rasnik, I., Cheng, W., Babcock, H.P., Gauss, G.H., Lohman, T.M. and Chu, S. (2002) Initiation and re-initiation of DNA unwinding by the Escherichia coli Rep helicase. Nature 419: 638–641.PubMedCrossRefGoogle Scholar
  24. Heimstadt, O. (1911) Das Fluoreszenzmicroskop. Z. wiss. Mikrosk. 28: 330.Google Scholar
  25. Kapanidis, A.N., Lee, N.K., Laurence, T.A., Doose, S., Margeat, E. and Weiss, S. (2004). Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules. Proc. Natl. Acad. Sci. USA 101: 8936–8941.PubMedCrossRefGoogle Scholar
  26. Kapanidis, A.N., Laurence, T.A., Lee, N.K., Margeat, E., Kong, X. and Weiss, S. (2005) Alternating-laser excitation of single molecules. Acc. Chem. Res. 38: 523–533.PubMedCrossRefGoogle Scholar
  27. Kask, P., Piksarv, P., Pooga, M., Mets, Ü. and Lippmaa, E. (1989) Separation of the rotational contribution in fluorescence correlation experiments. Biophys. J. 55: 213–220.CrossRefGoogle Scholar
  28. Kettling, U., Koltermann, A., Schwille, P. and Eigen, M. (1998) Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy. Proc. Natl. Acad. Sci. USA 95: 1416–1420.PubMedCrossRefGoogle Scholar
  29. Kohl, T., Heinze, K.G., Kuhlemann, R., Koltermann, A. and Schwille, P. (2002) A protease assay for two-photon crosscorrelation and FRET analysis based solely on fluorescent proteins. Proc. Natl. Acad. Sci. USA 99: 12161–12166.CrossRefGoogle Scholar
  30. Koppel, D.E. (1974) Statistical accuracy in fluorescence correlation spectroscopy. Phys. Rev. A. 10: 1938–1945.CrossRefGoogle Scholar
  31. Koppel, D.E., Axelrod, D., Schlessinger, J., Elson, E.L. and Webb, W.W. (1976) Dynamics of fluorescence marker concentration as a probe of mobility. Biophys. J. 16: 1315–1329.PubMedCrossRefGoogle Scholar
  32. Lamb, D.C., Schenk, A., Röcker, C. and Nienhaus, G.U. (2000a). Determining chemical rate coefficients using time-gated fluorescence correlation spectroscopy. J. Phys. Org. Chem. 13: 654–658.CrossRefGoogle Scholar
  33. Lamb, D.C., Schenk, A., Röcker, C., Scalfi-Happ, C. and Nienhaus, G.U. (2000b). Sensitivity enhancement in fluorescence correlation spectroscopy of multiple species using time-gated detection. Biophys. J. 79: 1129–1138.PubMedCrossRefGoogle Scholar
  34. Lee, N.K., Kapanidis, A.N., Wang, Y., Michalet, X., Mukhopadhyay, J., Ebright, R.H. and Weiss, S. (2005) Accurate FRET measurements within single diffusing biomolecules using alternating-laser excitation. Biophys. J. 88: 2939–2953.PubMedCrossRefGoogle Scholar
  35. Lehman, H. (1913) Das Lumineszenzmicroscop. Zeitschrift für Wissenschaftliche Microskopie 30: 417–470.Google Scholar
  36. Lu, H.P., Xun, L. and Xie, X.S. (1998) Single-molecule enzymatic dynamics. Science 282: 1877–1882.PubMedCrossRefGoogle Scholar
  37. Magde, D., Elson, E.L. and Webb, W.W. (1972) Thermodynamic fluctuations in a reacting system – measurement by fluorescence correlation spectroscopy. Phys. Rev. Lett. 29: 705–708.CrossRefGoogle Scholar
  38. Magde, D., Elson, E.L. and Webb, W.W. (1974) Fluorescence correlation spectroscopy. II. An experimental realization. Biopolymers 13: 29–61.PubMedCrossRefGoogle Scholar
  39. Magde, D. (1976) Chemical kinetics and fluorescence correlation spectroscopy. Q. Rev. Biophys. 9: 35–47.CrossRefGoogle Scholar
  40. Magde, D., Webb, W.W. and Elson, E.L. (1978) Fluorescence correlation spectroscopy. III. Uniform translation and laminar flow. Biopolymers 17: 361–376.CrossRefGoogle Scholar
  41. Meyer, T. and Schindler, H. (1988) Particle counting by fluorescence correlation spectroscopy. Simultaneous measurement of aggregation and diffusion of molecules in solutions and in membranes. Biophys. J. 54: 983–993.PubMedCrossRefGoogle Scholar
  42. Müller, B.K., Zaychikov, E., Bräuchle, C. and Lamb, D.C. (2005) Pulsed interleaved excitation. Biophys. J. 89: 3508–3522.PubMedCrossRefGoogle Scholar
  43. Müller, C.B., Loman, A., Pacheco, V., Koberling, F., Willbold, D., Richtering, W. and Enderlein, J. (2008) Precise measurement of diffusion by multi-color dual-focus fluorescence correlation spectroscopy. EPL 83: 46001.Google Scholar
  44. Petrasek, Z. and Schwille, P. (2008) Precise measurement of diffusion coefficients using scanning fluorescence correlation spectroscopy. Biophys. J. 94: 1437–1448.PubMedCrossRefGoogle Scholar
  45. Rauer, B., Neumann, E., Widengren, J. and Rigler, R. (1996) Fluorescence correlation spectrometry of the interaction kinetics of tetramethylrhodamin a-bungarotoxin with Torpedo californica acetylcholine receptor. Biophys. Chem. 58: 3–12.PubMedCrossRefGoogle Scholar
  46. Reichert, K. (1911) Das Fluorescenczmikroskop. Phys. Z. 12: 1010–1011.Google Scholar
  47. Rhoades, E., Gussakovsky, E. and Haran, G. (2003) Watching proteins fold one molecule at a time. Proc. Natl. Acad. Sci. USA 100: 3197–3202.PubMedCrossRefGoogle Scholar
  48. Ries, J. and Schwille, P. (2006) Studying slow membrane dynamics with continuous wave scanning fluorescence correlation spectroscopy. Biophys. J. 91: 1915–1924.PubMedCrossRefGoogle Scholar
  49. Ries, J., Chiantia, S. and Schwille, P. (2009) Accurate determination of membrane dynamics with line-scan FCS. Biophys. J. 96: 1999–2008.PubMedCrossRefGoogle Scholar
  50. Rigler, R., Kask, P., Mets, Ü. and Widengren, J. (1993) Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion. Eur. Biophys. J. 22: 169–175.CrossRefGoogle Scholar
  51. Schwille, P., Bieschke, J. and Oehlenschlager, F. (1997a). Kinetic investigations by fluorescence correlation spectroscopy: the analytical and diagnostic potential of diffusion studies. Biophys. Chem. 66: 211–228.PubMedCrossRefGoogle Scholar
  52. Schwille, P., MeyerAlmes, F.J. and Rigler, R. (1997b). Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution. Biophys. J. 72: 1878–1886.PubMedCrossRefGoogle Scholar
  53. Svedberg, T. and Inouye, K. (1911) Eine neue Methode zur Prüfung der Gültigkeit des Boyle-Gay-Lussacschen Gesetzes für Kolloide Lösungen. Z. Phys. Chem. 77: 145–191.Google Scholar
  54. Thompson, N.L., Burghardt, T.P. and Axelrod, D. (1981) Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy. Biophys. J. 33: 435–454.PubMedCrossRefGoogle Scholar
  55. Thompson, N.L. (1991) Fluorescence correlation spectroscopy. In: Topics in fluorescence spectroscopy, volume 1: techniques, J.R. Lakowicz, ed. Plenum Press, New York, NY, pp. 337–378.Google Scholar
  56. Torres, T. and Levitus, M. (2007) Measuring conformational dynamics: a new FCS-FRET approach. J. Phys. Chem. B 111: 7392–7400.PubMedCrossRefGoogle Scholar
  57. von Smoluchowski, M. (1906) Zur kinetischen Theorie dier Brownschen Molekularbewegung und der Suspensionen. Ann. Phys. 21: 756–780.CrossRefGoogle Scholar
  58. Webb, R.H. (1996) Confocal optical microscopy. Rep. Progr. Phys. 59: 427.CrossRefGoogle Scholar
  59. Wennmalm, S., Edman, L. and Rigler, R. (1997) Conformational fluctuations in single DNA molecules. Proc. Natl. Acad. Sci. USA 94: 10641–10646.PubMedCrossRefGoogle Scholar
  60. Widengren, J., Rigler, R. and Mets, Ü. (1994) Triplet-state monitoring by fluorescence correlation spectroscopy. J. Fluoresc. 4: 255–258.CrossRefGoogle Scholar
  61. Widengren, J., Mets, Ü. and Rigler, R. (1995) Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study. J. Phys. Chem. 99: 13368–13379.CrossRefGoogle Scholar
  62. Widengren, J., Schweinberger, E., Berger, S. and Seidel, C.A.M. (2001) Two new concepts to measure fluorescence resonance energy transfer via fluorescence correlation spectroscopy: theory and experimental realizations. J. Phys. Chem. A 105: 6851–6866.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  1. 1.Department for Chemistry and BiochemistryCenter for Nanoscience (CeNS) and Munich Center for Integrated Protein Science (CiPSM), Ludwig-Maximilians-Universität MünchenMunichGermany
  2. 2.Department for ChemistryCenter for Nanoscience (CeNS) and Munich Center for Integrated Protein Science (CiPSM), Ludwig-Maximilians-Universität MünchenMunichGermany
  3. 3.Department of PhysicsUniversity of Illinois at Urbana-ChampaignUrbanaUSA

Personalised recommendations