European Biophysics Journal

, Volume 34, Issue 4, pp 323–334 | Cite as

Global analysis of fluorescence fluctuation data

  • Victor V. Skakun
  • Mark A. Hink
  • Anatoli V. Digris
  • Ruchira Engel
  • Eugene G. Novikov
  • Vladimir V. Apanasovich
  • Antonie J. W. G. VisserEmail author


Over the last decade the number of applications of fluorescence correlation spectroscopy (FCS) has grown rapidly. Here we describe the development and application of a software package, FCS Data Processor, to analyse the acquired correlation curves. The algorithms combine strong analytical power with flexibility in use. It is possible to generate initial guesses, link and constrain fit parameters to improve the accuracy and speed of analysis. A global analysis approach, which is most effective in analysing autocorrelation curves determined from fluorescence fluctuations of complex biophysical systems, can also be implemented. The software contains a library of frequently used models that can be easily extended to include user-defined models. The use of the software is illustrated by analysis of different experimental fluorescence fluctuation data sets obtained with Rhodamine Green in aqueous solution and enhanced green fluorescent protein in vitro and in vivo.


Fluorescence correlation spectroscopy Global analysis Green fluorescent protein Translational diffusion Triplet state 



Enhanced green fluorescent protein


Fluorescence correlation spectroscopy


Fluorescence cross-correlation spectroscopy


Green fluorescent protein


Phosphate-buffered saline


  1. Akcakir O, Therrien J, Belomoin G, Barry N, Müller JD, Gratton E, Nayfeh M (2000) Detection of luminescent single ultra-small silicon nanoparticles using fluorescence fluctuation spectroscopy. Appl Phys Lett 76:1857–1859CrossRefGoogle Scholar
  2. Aragón SR, Pecora R (1975) Fluorescence correlation spectroscopy and Brownian rotational diffusion. Biopolymers 14:119–138Google Scholar
  3. Aragón SR, Pecora R (1976) Fluorescence correlation spectroscopy as a probe of molecular dynamics. J Chem Phys 64:1791–1803CrossRefGoogle Scholar
  4. Bacia K, Schwille P (2003) A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy. Methods 29:74–85CrossRefGoogle Scholar
  5. Bacia K, Majoul IV, Schwille P (2002) Probing the endocytic pathway in live cells using dual-color fluorescence cross-correlation analysis. Biophys J 83:1184–1193Google Scholar
  6. Bastiaens PIH, Pap EHW, Widengren J, Rigler R, Visser AJWG (1994) Fluorescence methods to study lipid-protein association: the interaction of protein kinase C with lipid-loaded mixed micelles. J Fluoresc 4: 377–383Google Scholar
  7. Beechem JM (1992) Global analysis of biochemical and biophysical data. Methods Enzymol 210:37–54Google Scholar
  8. Beechem JM, Gratton E, Ameloot M, Knutson JR, Brand L (1991) The global analysis of fluorescence intensity and anisotropy decay data: second-generation theory and programs. In: Lakowicz JR (ed) Topics in fluorescence spectroscopy, vol 2. Plenum, New York, pp 241–305Google Scholar
  9. Berland KM, So PTC, Gratton E (1995) Two-photon fluorescence correlation spectroscopy: method and application to the cellular environment. Biophys J 68:694–701Google Scholar
  10. Bevington PR (1969) Data reduction and error analysis for the physical sciences. McGraw-Hill, New YorkGoogle Scholar
  11. Bonnet G, Krichevsky O, Libchaber A (1998) Kinetics of conformational fluctuations in DNA hairpin-loops. Proc Natl Acad Sci USA 95:8602–8606CrossRefGoogle Scholar
  12. Boonen G, Pramanik A, Rigler R, Häberlein H (2000) Evidence for specific interactions between a kavain derivative and human cortical neurons measured by fluorescence correlation spectroscopy. Planta Med 66:7–10CrossRefGoogle Scholar
  13. Brock R, Hink MA, Jovin TM (1998) Fluorescence correlation microscopy of cells in the presence of autofluorescence. Biophys J 75:2547–2557Google Scholar
  14. Brock R, Vamosi G, Vereb G, Jovin TM (1999) Rapid characterization of green fluorescent protein fusion proteins on the molecular and cellular level by fluorescence correlation microscopy. Proc Natl Acad Sci USA 96:10123–10128CrossRefGoogle Scholar
  15. Chen Y, Müller JD, So PTC, Gratton E (1999) The photon counting histogram in fluorescence fluctuation spectroscopy. Biophys J 77:553–567Google Scholar
  16. Chen Y, Müller JD, Ruan Q, Gratton E (2002) Molecular brightness characterization of EGFP in vivo by fluorescence fluctuation spectroscopy. Biophys J 82:133–144Google Scholar
  17. Cluzel P, Surette M, Leibler S (2000) An ultrasensitive bacterial motor revealed by monitoring signaling proteins in single cells. Science 287:1652–1655CrossRefGoogle Scholar
  18. Di Cera E (1992) Use of weighting functions in data fitting. Methods Enzymol 210:68–87Google Scholar
  19. Edman L, Mets Ü, Rigler R (1996) Conformational transitions monitored for single molecules in solution. Proc Natl Acad Sci USA 93:6710–6715CrossRefGoogle Scholar
  20. Eggeling C, Berger S, Brand L, Fries JR, Schaffer J, Volkmer A, Seidel CAM (2001) Data registration and selective single-molecule analysis using multi-parameter fluorescence detection. J Biotechnol 86:163–180CrossRefGoogle Scholar
  21. Ehrenberg M, Rigler R (1974) Rotational Brownian motion and fluorescence intensity fluctuations. Chem Phys 4:390–401CrossRefGoogle Scholar
  22. Eid JS, Müller JD, Gratton E (2000) Data acquisition card for fluctuation correlation spectroscopy allowing full access to the detected photon sequence. Rev Sci Instrum 71:361–368CrossRefGoogle Scholar
  23. Eigen M, Rigler R (1994) Sorting single molecules: application to diagnostics and evolutionary biotechnology. Proc Natl Acad Sci USA 91:5740–5747Google Scholar
  24. Elson EL, Magde D (1974) Fluorescence correlation spectroscopy I Conceptual basis and theory. Biopolymers 13:1–27CrossRefGoogle Scholar
  25. Firtel RA, Chung CY (2000) The molecular genetics of chemotaxis: sensing and responding to chemoattractant gradients. Bioessays 22:603–615Google Scholar
  26. Fradin C, Abu-Arish A, Granek R, Elbaum M (2003) Fluorescence correlation spectroscopy close to a fluctuating membrane. Biophys J 84:2005–2020Google Scholar
  27. Goedhart J, Röhrig H, Hink MA, van Hoek A, Visser AJWG, Bisseling T, Gadella Jr TWJ (1999) Nod factors integrate spontaneously in biomembranes and transfer rapidly between membranes and to root hairs, but transbilayer flip-flop does not occur. Biochemistry 38:10898–10907CrossRefGoogle Scholar
  28. Goedhart J, Hink MA, Visser AJWG, Bisseling T, Gadella TWJ (2000) In vivo fluorescence correlation microscopy (FCM) reveals accumulation and immobilization of Nod factors in root hair cell walls. Plant J 21:109–119CrossRefGoogle Scholar
  29. Haupts U, Maiti S, Schwille P, Webb WW (1998) Dynamics of fluorescence fluctuations in green fluorescent protein observed by fluorescence correlation spectroscopy. Proc Natl Acad Sci USA 95:573–578CrossRefGoogle Scholar
  30. Heikal AA, Hess ST, Baird GS, Tsien RY, Webb WW (2000) Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: coral red (dsRed) and yellow (Citrine). Proc Natl Acad Sci USA 97:11996–12001CrossRefGoogle Scholar
  31. Heinze KG, Koltermann A, Schwille P (2000) Simultaneous two-photon excitation of distinct labels for dual-color fluorescence crosscorrelation analysis. Proc Natl Acad Sci USA 97:10377–10382CrossRefGoogle Scholar
  32. Henriksson M, Pramanik A, Shafqat J, Zhong Z, Tally M, Ekberg K, Wahren J, Rigler R, Johansson J, Jörnvall H (2001) Specific binding of proinsulin C-peptide to intact and to detergent-solubilized human skin fibroblasts. Biochem Biophys Res Commun 280:423–427CrossRefGoogle Scholar
  33. Hess ST, Huang S, Heikal AA, Webb WW (2002) Biological and chemical applications of fluorescence correlation spectroscopy: a review. Biochemistry 41:697–705CrossRefGoogle Scholar
  34. Hink MA, Visser AJWG (1998) Characterization of membrane mimetic systems with fluorescence correlation spectroscopy. In: Rettig W, Strehmel B, Schrader S (eds) Applied fluorescence in chemistry, biology and medicine. Springer, Berlin Heidelberg New York, pp 101–118Google Scholar
  35. Hink MA, van Hoek A, Visser AJWG (1999) Dynamics of phospholipid molecules in micelles: characterization with fluorescence correlation spectroscopy and time-resolved fluorescence anisotropy. Langmuir 15:992–997CrossRefGoogle Scholar
  36. Hink MA, Griep RA, Borst JW, van Hoek A, Eppink MHM, Schots A, Visser AJWG (2000) Structural dynamics of green fluorescent protein alone and fused with a single chain Fv protein. J Biol Chem 275:17556–17560CrossRefGoogle Scholar
  37. Hink MA, Borst JW, Visser AJWG (2003) Fluorescence correlation spectroscopy of GFP fusion proteins in living plant cells. Methods Enzymol 361:93–112Google Scholar
  38. Kask P, Palo K, Ullmann D, Gall K (1999) Fluorescence-intensity distribution analysis and its application in biomolecular detection technology. Proc Natl Acad Sci USA 96:13756–13761CrossRefGoogle Scholar
  39. Kettling U, Koltermann A, Schwille P, Eigen M (1998) Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy. Proc Natl Acad Sci USA 95:1416–1420CrossRefGoogle Scholar
  40. Köhler RH, Schwille P, Webb WW, Hanson MR (2000) Active protein transport through plastid tubules: velocity quantified by fluorescence correlation spectroscopy. J Cell Sci 113:3921–3930Google Scholar
  41. Koltermann A, Kettling U, Bieschke J, Winkler T, Eigen M (1998) Rapid assay processing by integration of dual-color fluorescence cross-correlation spectroscopy: high-throughput screening for enzyme activity. Proc Natl Acad Sci USA 95:1421–1426CrossRefGoogle Scholar
  42. Koopman WJH, Hink MA, Visser AJWG, Roubos EW, Jenks BG (1999) Evidence that Ca2+ -waves in Xenopus melanotropes depend on calcium-induced calcium release: a fluorescence correlation microscopy and linescanning study. Cell Calcium 26:59–67CrossRefGoogle Scholar
  43. Koppel DE (1974) Statistical accuracy in fluorescence correlation spectroscopy. Phys Rev A 10:1938–1945CrossRefGoogle Scholar
  44. Korlach J, Schwille P, Webb W W, Feigenson G W (1999) Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy. Proc Natl Acad Sci USA 96:8461–8466CrossRefGoogle Scholar
  45. Kunst, BH, Schots A, Visser AJWG (2002) Detection of flowing fluorescent particles in a microcapillary using fluorescence correlation spectroscopy. Anal Chem 74:5350–5357CrossRefGoogle Scholar
  46. LaClair JJ (1997) Selective detection of the carbohydrate-bound state of concanvalin A at the single molecule level. J Amer Chem Soc 119:7676–7684CrossRefGoogle Scholar
  47. 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
  48. Magde D, Elson EL, Webb WW (1974) Fluorescence correlation spectroscopy. II. An experimental realization. Biopolymers 13:29–61CrossRefGoogle Scholar
  49. Magde D, Webb WW, Elson EL (1977) Fluorescence correlation spectroscopy III uniform translation and laminar flow. Biopolymers 17:377–412Google Scholar
  50. Maiti S, Haupts U, Webb WW (1997) Fluorescence correlation spectroscopy: diagnostics for sparse molecules. Proc Natl Acad Sci USA 94:11753–11757CrossRefGoogle Scholar
  51. Marquardt DW (1963) An algorithm for least squares estimation of nonlinear parameters. J Soc Indust Appl Math 11:431–441Google Scholar
  52. Meseth U, Wohland T, Rigler R, Vogel, H (1999) Resolution of fluorescence correlation measurements. Biophys J 76:1619–1631Google Scholar
  53. Novikov EG, van Hoek A, Visser AJWG, Hofstraat JW (1999) Linear algorithms for stretched exponential decay analysis. Opt Commun 166:189–199CrossRefGoogle Scholar
  54. Oehlenschläger F, Schwille P, Eigen M (1996) Detection of HIV-1 RNA by nucleic acid sequence-based amplification combined with fluorescence correlation spectroscopy. Proc Natl Acad Sci USA 93:12811–12816CrossRefGoogle Scholar
  55. Palmer AG, Thompson NL (1987) Molecular aggregation characterized by high order autocorrelation in fluorescence correlation spectroscopy. Biophys J 52:257–270Google Scholar
  56. Parent CA, Devreotes PN (1999) A cell’s sense of direction. Science 284:765–770CrossRefGoogle Scholar
  57. Politz JC, Brown ES, Wolf DE, Pederson T (1998) Intranuclear diffusion and hybridisation state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells. Proc Natl Acad Sci USA 95:6043–6048CrossRefGoogle Scholar
  58. Pramanik A, Juréus A, Langel Ü, Bartfai T, Rigler R (1999) Galanin receptor binding studies in the membranes of cultured cells measured by fluorescence correlation spectroscopy. Biomed Chromatogr 13:119–120CrossRefGoogle Scholar
  59. Qian H, Elson EL (1991) Analysis of confocal laser-microscope optics for 3-D fluorescence correlation spectroscopy. Appl Opt 30:1185–1195Google Scholar
  60. Rarbach M, Kettling U, Koltermann K, Eigen M (2001) Dual color fluorescence cross-correlation spectroscopy for monitoring the kinetics of enzyme-catalyzed reactions. Methods 24:104–116CrossRefGoogle Scholar
  61. Rauer B, Neumann E, Widengren J, Rigler R (1996) Fluorescence correlation spectroscopy of the interaction kinetics of tetramethylrhodamin α-bungarotoxin with Torpedo calnifornica acetylcholine receptor. Biophys Chem 58:3–12CrossRefGoogle Scholar
  62. Rigler R (1995) Fluorescence correlations, single molecule detection and large number screening, applications in biotechnology. J Biotechnol 41:177–186CrossRefGoogle Scholar
  63. Rigler R and Elson EL (eds) (2001) Fluorescence correlation spectroscopy, theory and applications. Springer, Berlin Heidelberg New YorkGoogle Scholar
  64. Rigler R, Mets Ü, Widengren J, Kask P (1993) Fluorescence correlation spectroscopy with high count rates and low background, analysis of translational diffusion. Eur Biophys J 22:169–175CrossRefGoogle Scholar
  65. Rigler R, Pramanik A, Jonasson P, Kratz G, Jansson OT, Nygren PÅ, Ståhl S, Ekberg K, Johansson BL, Uhlén S, Uhlén M, Jörnvall H, Wahren J (1999a) Specific binding of proinsulin C-peptide to human cell membranes. Proc Natl Acad Sci USA 96:13318–13323CrossRefGoogle Scholar
  66. Rigler R, Földes-Papp Z, Meyer-Almes FJ, Sammet C, Volcker M, Schnetz A (1999b) Fluorescence cross-correlation: a new concept for polymerase chain reaction. J Biotechnol 63:97–109CrossRefGoogle Scholar
  67. Rippe K (2000) Simultaneous binding of two DNA duplexes to the NtrC-enhancer complex studied by two-color fluorescence cross-correlation spectroscopy. Biochemistry 39:2131–2139CrossRefGoogle Scholar
  68. Ruchira, Hink MA, Bosgraaf L, Van Haastert PJM, Visser AJWG (2004) Pleckstrin homology domain diffusion in Dictyostelium cytoplasm studied using fluorescence correlation spectroscopy. J Biol Chem 279:10013–10019CrossRefGoogle Scholar
  69. Saffarian S, Elson EL (2003) Statistical analysis of fluorescence correlation spectroscopy: the standard deviation and bias. Biophys J 84:2030–2042Google Scholar
  70. Schenk A, Ivanchenko S, Röcker C, Wiedenmann J, Nienhaus GU (2004) Photodynamics of red fluorescent proteins studied by fluorescence correlation spectroscopy. Biophys J 86: 384–394Google Scholar
  71. Schwille P (2001a) Fluorescence correlation spectroscopy and its potential for intracellular applications. Cell Biochem Biophys 34:383–408CrossRefGoogle Scholar
  72. Schwille P (2001b) Cross-correlation analysis in FCS. In: Rigler R and Elson EL (eds) Fluorescence correlation spectroscopy. Theory and applications. Springer, Berlin Heidelberg New York, pp 360–378Google Scholar
  73. Schwille P, Meyer-Almes FJ, Rigler R (1997) Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution. Biophys J 72:1878–1886Google Scholar
  74. Schwille P, Korlach J, Webb WW (1999a) Fluorescence correlation spectroscopy with single molecule sensitivity on cell and model membranes. Cytometry 36:176–182CrossRefGoogle Scholar
  75. Schwille P, Haupts U, Maiti S, Webb WW (1999b) Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation. Biophys J 77:2251–2265Google Scholar
  76. Schwille P, Kummer S, Heikal AA, Moerner WE, Webb WW (2000) Fluorescence correlation spectroscopy reveals fast optical excitation-driven intramolecular dynamics of yellow fluorescent proteins excitation. Proc Natl Acad Sci USA 97:151–156CrossRefGoogle Scholar
  77. Thompson NL (1991) Fluorescence correlation spectroscopy. In: Lakowicz JR (ed) Topics in fluorescence spectroscopy. Plenum, New York, pp 337–378Google Scholar
  78. Thompson NL, Lieto AM, Allen NW (2002) Recent advances in fluorescence correlation spectroscopy. Curr Opinion Struct Biol 12:634–641CrossRefGoogle Scholar
  79. Van Craenenbroeck E, Vercammen J, Matthys G, Beirlent J, Marot C, Hoebeke J, Strobbe R, Engelborghs Y (2001) Heuristic statistical analysis of fluorescence fluctuation data with bright spikes: application to ligand binding to the human serotonin receptor expressed in Escherichia coli cells. Biol Chem 382:355–361CrossRefGoogle Scholar
  80. Van den Berg PAW, Widengren J, Hink MA, Rigler R, Visser AJWG (2001) Fluorescence correlation spectroscopy of flavins and flavoenzymes: photochemical and photophysical aspects. Spectrochim Acta 57A:2135–2144Google Scholar
  81. Verveer PJ, Squire A, Bastiaens PIH (2000) Global analysis of fluorescence lifetime imaging microscopy data. Biophys J 84:2127–2137Google Scholar
  82. Visser NV, Hink MA, Van Hoek A, Visser AJWG (1999) Comparison between fluorescence correlation spectroscopy and time-resolved fluorescence anisotropy as illustrated with a fluorescent dextran conjugate. J Fluoresc 9:251–255CrossRefGoogle Scholar
  83. Visser AJWG, Hink MA (1999) New perspectives of fluorescence correlation spectroscopy. J Fluoresc 9:81–87Google Scholar
  84. Visser AJWG, Van den Berg PAW, Hink MA, Petushkov VN (2001) Fluorescence correlation spectroscopy of flavins and flavoproteins. In: Rigler R, Elson EL (eds) Fluorescence correlation spectroscopy, theory and applications. Springer, Berlin Heidelberg New York, pp 9–24Google Scholar
  85. Wachsmuth M, Waldeck W, Langowski J (2000) Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy. J Mol Biol 298:677–689CrossRefGoogle Scholar
  86. Walter NS, Schwille P, Eigen M (1996) Fluorescence correlation analysis of probe diffusion simplifies quantitative pathogen detection by PCR. Proc Natl Acad Sci USA 93:12805–12810CrossRefGoogle Scholar
  87. Widengren J, Rigler R (1998) Fluorescence correlation spectroscopy as a tool to investigate chemical reactions in solutions and on cell surfaces. Cell Mol Biol 44:857–879Google Scholar
  88. Widengren J, Mets Ü, Rigler R (1995) Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study. J Phys Chem 99:13368–13379Google Scholar
  89. Wohland T, Rigler R, Vogel H (2001) The standard deviation in fluorescence correlation spectroscopy. Biophys J 80:2987–2999Google Scholar

Copyright information

© EBSA 2005

Authors and Affiliations

  • Victor V. Skakun
    • 1
  • Mark A. Hink
    • 2
  • Anatoli V. Digris
    • 1
  • Ruchira Engel
    • 2
  • Eugene G. Novikov
    • 3
  • Vladimir V. Apanasovich
    • 1
  • Antonie J. W. G. Visser
    • 2
    Email author
  1. 1.Department of Systems AnalysisBelarusian State UniversityMinskBelarus
  2. 2.MicroSpectroscopy Centre, Laboratory of BiochemistryWageningen UniversityET WageningenThe Netherlands
  3. 3.Institut Curie, Section de RechercheParisFrance

Personalised recommendations