European Biophysics Journal

, Volume 41, Issue 12, pp 1055–1064 | Cite as

Simultaneous diffusion and brightness measurements and brightness profile visualization from single fluorescence fluctuation traces of GFP in living cells

  • Victor V. SkakunEmail author
  • Ruchira Engel
  • Jan Willem Borst
  • Vladimir V. Apanasovich
  • Antonie J. W. G. Visser
Original Paper


Fluorescence correlation spectroscopy (FCS) and photon-counting histogram (PCH) analysis use the same experimental fluorescence intensity fluctuations, but each analytical method focuses on a different property of the signal. The time-dependent decay of the correlation of fluorescence fluctuations is measured in FCS yielding, for instance, molecular diffusion coefficients. The amplitude distribution of these fluctuations is calculated by PCH analysis yielding information about the molecular brightness of fluorescent species. Analysis of both FCS and PCH results in the molecular concentration of the sample. Using a previously described global analysis procedure we report here precise, simultaneous measurements of diffusion constants and brightness values from single fluorescence fluctuation traces of green-fluorescent protein (GFP, S65T) in the cytoplasm of Dictyostelium cells. The use of a polynomial profile in PCH analysis, describing the detected three-dimensional shape of the confocal volume, enabled us to obtain well fitting results for GFP in cells. We could visualize the polynomial profile and show its deviation from a Gaussian profile.


Green fluorescent protein Fluorescence correlation spectroscopy Photon-counting histogram analysis Fluorescence intensity distribution analysis Global analysis of ACF and PCD Dictyostelium cells 

Supplementary material

249_2012_864_MOESM1_ESM.doc (741 kb)
Supplementary material 1 (DOC 741 kb)


  1. Al-Soufi W, Reija B, Felekyan S, Seidel CAM, Novo M (2008) Dynamics of supramolecular association monitored by fluorescence correlation spectroscopy. Chem Phys Chem 9:1819–1827PubMedCrossRefGoogle Scholar
  2. Bacia K, Kim SA, Schwille P (2006) Fluorescence cross-correlation spectroscopy in living cells. Nat Methods 3:83–89PubMedCrossRefGoogle Scholar
  3. 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 96:10123–10128PubMedCrossRefGoogle Scholar
  4. Chen Y, Müller JD, So PT, Gratton E (1999) The photon-counting histogram in fluorescence fluctuation spectroscopy. Biophys J 77:553–567PubMedCrossRefGoogle Scholar
  5. Chen Y, Müller JD, Tetin SY, Tyner JD, Gratton E (2000) Probing ligand protein binding equilibria with fluorescence fluctuation spectroscopy. Biophys J 79:1074–1084PubMedCrossRefGoogle Scholar
  6. Chen Y, Müller JD, Ruan Q, Gratton E (2002) Molecular brightness characterization of eGFP in vivo by fluorescence fluctuation spectroscopy. Biophys J 82:133–144PubMedCrossRefGoogle Scholar
  7. Chen Y, Wei L-N, Müller JD (2003) Probing protein oligomerization in living cells with fluorescence fluctuation spectroscopy. Proc Natl Acad Sci USA 100:15492–15497PubMedCrossRefGoogle Scholar
  8. Elson EL, Magde D (1974) Fluorescence correlation spectroscopy. I Conceptual basis and theory. Biopolymers 13:1–27CrossRefGoogle Scholar
  9. Enderlein J, Gregor I, Patra D, Fitter J (2004) Art and artefacts of fluorescence correlation spectroscopy. Curr Pharm Biotech 5:155–161CrossRefGoogle Scholar
  10. Enderlein J, Gregor I, Patra D, Dertinger T, Kaupp UB (2005) Performance of fluorescence correlation spectroscopy for measuring diffusion and concentration. Chem Phys Chem 6:2324–2336PubMedCrossRefGoogle Scholar
  11. Gopich V, Szabo A (2005) Photon-counting histograms for diffusive fluorophores. J Phys Chem B 109:17683–17688PubMedCrossRefGoogle Scholar
  12. Haustein E, Schwille P (2007) Fluorescence correlation spectroscopy: novel variations of an established technique. Annu Rev Biophys Biomol Struct 36:151–169PubMedCrossRefGoogle Scholar
  13. Hess ST, Huang S, Heikal AA, Webb WW (2002) Biological and chemical applications of fluorescence correlation spectroscopy: a review. Biochemistry 41:697–705PubMedCrossRefGoogle Scholar
  14. Hink MA, Shah K, Russinova E, de Vries SC, Visser AJWG (2008) Fluorescence fluctuation analysis of Arabidopsis thaliana somatic embryogenesis receptor-like kinase and brassinosteroid insensitive 1 receptor oligomerization. Biophys J 94:1052–1062PubMedCrossRefGoogle Scholar
  15. Huang B, Perroud TD, Zare RN (2004) Photon-counting histogram: one-photon excitation. Chem Phys Chem 5:1523–1531PubMedCrossRefGoogle Scholar
  16. 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 96:13756–13761PubMedCrossRefGoogle Scholar
  17. Kask P, Palo K, Fay N, Brand L, Mets U, Ullmann D, Jungmann J, Pschorr J, Gall K (2000) Two-dimensional fluorescence intensity distribution analysis: theory and applications. Biophys J 78:1703–1713PubMedCrossRefGoogle Scholar
  18. Knol JC, Engel R, Blaauw M, Visser AJ, van Haastert PJM (2005) The phosducin-like protein PhLP1 is essential for Gβγ dimer formation in Dictyostelium discoideum. Mol Cell Biol 25:8393–8400PubMedCrossRefGoogle Scholar
  19. Krichevsky O, Bonnet G (2002) Fluorescence correlation spectroscopy: the technique and its applications. Rep Prog Phys 65:251–297CrossRefGoogle Scholar
  20. Magde D, Elson EL, Webb WW (1972) Thermodynamic fluctuations in a reaction system: measurement by fluorescence correlation spectroscopy. Phys Rev Lett 29:705–708CrossRefGoogle Scholar
  21. Magde D, Elson EL, Webb WW (1974) Fluorescence correlation spectroscopy II. An experimental realization. Biopolymers 13:29–61PubMedCrossRefGoogle Scholar
  22. Malikova NP, Visser NV, van Hoek A, Skakun VV, Vysotski ES, Lee J, Visser AJ (2011) Green-fluorescent protein from the bioluminescent jellyfish Clytia gregaria is an obligate dimer and does not form a stable complex with the Ca2+-discharged photoprotein clytin. Biochemistry 50:4232–4241PubMedCrossRefGoogle Scholar
  23. Meng F, Ma H (2006) A comparison between photon-counting histogram and fluorescence intensity distribution analysis. J Phys Chem B 110:25716–25720PubMedCrossRefGoogle Scholar
  24. Müller JD, Chen Y, Gratton E (2000) Resolving heterogeneity on the single molecular level with the photon-counting histogram. Biophys J 78:474–486PubMedCrossRefGoogle Scholar
  25. Müller CB, Loman A, Pacheco V, Koberling F, Willbold D, Richtering W, Enderlein J (2008) Precise measurement of diffusion by mult-color dual-focus fluorescence correlation spectroscopy. Eur Phys Lett 83:46001CrossRefGoogle Scholar
  26. Palo K, Metz U, Jager S, Kask P, Gall K (2000) Fluorescence intensity multiple distributions analysis: concurrent determination of diffusion times and molecular brightness. Biophys J 79:2858–2866PubMedCrossRefGoogle Scholar
  27. Palo K, Mets U, Loorits V, Kask P (2006) Calculation of photon count number distributions via master equations. Biophys J 90:2179–2191PubMedCrossRefGoogle Scholar
  28. Perroud TD, Huang B, Zare RN (2005) Effect of bin time on the photon-counting histogram for one-photon excitation. Chem Phys Chem 6:905–912PubMedCrossRefGoogle Scholar
  29. Rigler R, Elson ES (eds) (2001) Fluorescence correlation spectroscopy. Theory and applications. Springer, BerlinGoogle Scholar
  30. Rigler R, Mets U, 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
  31. Ruchira, Hink MA, Bosgraaf L, van Haastert PJ, Visser AJ (2004) Pleckstrin homology domain diffusion in Dictyostelium cytoplasm studied using fluorescence correlation spectroscopy. J Biol Chem 279:10013–10019Google Scholar
  32. Skakun VV, Hink MA, Digris AV, Engel R, Novikov EG, Apanasovich VV, Visser AJ (2005) Global analysis of fluorescence fluctuation data. Eur Biophys J 34:323–334PubMedCrossRefGoogle Scholar
  33. Skakun VV, Engel R, Digris AV, Borst JW, Visser AJ (2011) Global analysis of autocorrelation functions and photon-counting distributions. Front Biosci (Elite Ed) 3:489–505CrossRefGoogle Scholar
  34. Thompson NL, Lieto AM, Allen NW (2002) Recent advances in fluorescence correlation spectroscopy. Curr Opin Struct Biol 12:634–641PubMedCrossRefGoogle Scholar
  35. Widengren J, Mets U, Rigler R (1995) Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study. J Phys Chem 99:13368–13379CrossRefGoogle Scholar

Copyright information

© European Biophysical Societies' Association 2012

Authors and Affiliations

  • Victor V. Skakun
    • 1
    Email author
  • Ruchira Engel
    • 2
    • 3
  • Jan Willem Borst
    • 2
  • Vladimir V. Apanasovich
    • 1
  • Antonie J. W. G. Visser
    • 2
  1. 1.Department of Systems Analysis and Computer SimulationBelarusian State UniversityMinskBelarus
  2. 2.Laboratory of Biochemistry, and Microspectroscopy CentreWageningen UniversityWageningenThe Netherlands
  3. 3.Department of ImmunopathologySanquin Blood Supply FoundationAmsterdamThe Netherlands

Personalised recommendations