European Biophysics Journal

, Volume 38, Issue 6, pp 813–828 | Cite as

Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy

Original Paper


The spatial and temporal fluctuation microscope (STFM) presented here extends the concept of a fluorescence confocal laser scanning microscope to illumination and detection along a line. The parallel multichannel acquisition of the fluorescence signal was accomplished by using a single line of an electron-multiplying charge-coupled device camera at 14 μs time resolution for detection of the fluorescence signal. The STFM system provided fast confocal imaging (30 images per second) and allowed for the spatially resolved detection of particle concentration fluctuations in fluorescence correlation spectroscopy experiments. For the application of the STFM, an approximated theoretical description of the beam geometry, the point-spread function, and the fluorescence auto- and cross-correlation functions were derived. The STFM was applied to studies of the dynamics of promyelocytic leukemia nuclear bodies, green fluorescent protein, and chromatin-remodeling complexes in living cells. The results demonstrate the unique capabilities of the STFM for characterizing the position-dependent translocations and interactions of proteins in the cell.


Fluorescence correlation spectroscopy Single-particle tracking Promyelocytic leukemia nuclear body Green fluorescent protein Chromatin-remodeling complex Protein dynamics in living cells 



Green fluorescent protein


Mean-squared displacement


Single-particle tracking


Fluorescence recovery after photobleaching


Fluorescence correlation spectroscopy


Fluorescence cross-correlation spectroscopy


Spatial and temporal fluctuation microscope/microscopy


Fluorescence confocal laser-scanning microscope


Electron-multiplying charge-coupled device


Promyelocytic leukemia nuclear body


Point-spread function


Total internal reflection fluorescence


  1. Axelrod D, Koppel DE, Schlessinger J, Elson E, Webb WW (1976) Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J 16:1055–1069PubMedCrossRefGoogle Scholar
  2. Bayer J, Radler JO (2006) DNA microelectrophoresis using double focus fluorescence correlation spectroscopy. Electrophoresis 27:3952–3963PubMedCrossRefGoogle Scholar
  3. Beaudouin J, Mora-Bermúdez F, Klee T, Daigle N, Ellenberg J (2006) Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins. Biophys J 90:1878–1894PubMedCrossRefGoogle Scholar
  4. Becker PB, Horz W (2002) ATP-dependent nucleosome remodeling. Annu Rev Biochem 71:247–273PubMedCrossRefGoogle Scholar
  5. Berland KM, So PT, Gratton E (1995) Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment. Biophys J 68:694–701PubMedCrossRefGoogle Scholar
  6. Berland KM, So PTC, Chen Y, Mantulin WW, Gratton E (1996) Scanning 2-photon fluctuation correlation spectroscopy—particle counting measurements for detection of molecular aggregation. Biophys J 71:410–420PubMedCrossRefGoogle Scholar
  7. Borlinghaus RT (2006) High speed scanning has the potential to increase fluorescence yield and to reduce photobleaching. Micr Res Tech 69:689–692CrossRefGoogle Scholar
  8. Brinkmeier M, Dorre K, Riebeseel K, Rigler R (1997) Confocal spectroscopy in microstructures. Biophys Chem 66:229–239PubMedCrossRefGoogle Scholar
  9. Brinkmeier M, Dorre K, Stephan J, Eigen M (1999) Two beam cross correlation: a method to characterize transport phenomena in micrometer-sized structures. Anal Chem 71:609–616CrossRefGoogle Scholar
  10. Brown CM, Dalal RB, Hebert B, Digman MA, Horwitz AR, Gratton E (2008) Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope. J Microsc 229:78–91PubMedCrossRefGoogle Scholar
  11. Burkhardt M, Schwille P (2006) Electron multiplying CCD based detection for spatially resolved fluorescence correlation spectroscopy. Opt Exp 14:5013–5020CrossRefGoogle Scholar
  12. Cairns BR (2007) Chromatin remodeling: insights and intrigue from single-molecule studies. Nat Struct Mol Biol 14:989–996PubMedCrossRefGoogle Scholar
  13. Cole NB, Smith CL, Sciaky N, Terasaki M, Edidin M, Lippincott-Schwartz J (1996) Diffusional mobility of Golgi proteins in membranes of living cells. Science 273:797–801PubMedCrossRefGoogle Scholar
  14. Collins N, Poot RA, Kukimoto I, Garcia-Jimenez C, Dellaire G, Varga-Weisz PD (2002) An ACF1-ISWI chromatin-remodeling complex is required for DNA replication through heterochromatin. Nat Genet 32:627–632PubMedCrossRefGoogle Scholar
  15. Cutts LS, Roberts PA, Adler J, Davies MC, Melia CD (1995) Determination of localized diffusion coefficients in gels using confocal scanning laser microscopy. J Microsc 180:131–139Google Scholar
  16. Digman MA, Brown CM, Sengupta P, Wiseman PW, Horwitz AR, Gratton E (2005a) Measuring fast dynamics in solutions and cells with a laser scanning microscope. Biophys J 89:1317–1327PubMedCrossRefGoogle Scholar
  17. Digman MA, Sengupta P, Wiseman PW, Brown CM, Horwitz AR, Gratton E (2005b) Fluctuation correlation spectroscopy with a laser-scanning microscope: exploiting the hidden time structure. Biophys J 88:L33–L36PubMedCrossRefGoogle Scholar
  18. Dusch E, Dorval T, Vincent N, Wachsmuth M, Genovesio A (2007) Three-dimensional point spread function model for line-scanning confocal microscope with high-aperture objective. J Microsc 228:132–138PubMedCrossRefGoogle Scholar
  19. Elson EL, Magde D (1974) Fluorescence correlation spectroscopy. I: Conceptual basis and theory. Biopolymers 13:1–27CrossRefGoogle Scholar
  20. Gomez DE, Califano M, Mulvaney P (2006) Optical properties of single semiconductor nanocrystals. Phys Chem Chem Phys 8:4989–5011PubMedCrossRefGoogle Scholar
  21. Görisch SM, Wachsmuth M, Ittrich C, Bacher CP, Rippe K, Lichter P (2004) Nuclear body movement is determined by chromatin accessibility and dynamics. Proc Natl Acad Sci USA 101:13221–13226PubMedCrossRefGoogle Scholar
  22. Görisch SM, Lichter P, Rippe K (2005) Mobility of multi-subunit complexes in the nucleus: chromatin dynamics and accessibility of nuclear subcompartments. Histochem Cell Biol 123:217–228PubMedCrossRefGoogle Scholar
  23. Gunkel M, Erdel F, Rippe K, Lemmer P, Kaufmann R, Hörmann C, Amberger R, Cremer C (2009) Dual color localization microscopy of cellular nanostructures. Biotechnol J (in press)Google Scholar
  24. Hecht E (1989) Optics. Addisson-Wesley, Longmann, New YorkGoogle Scholar
  25. Hess ST, Webb WW (2002) Focal volume optics and experimental artifacts in confocal fluorescence correlation spectroscopy. Biophys J 83:2300–2317PubMedCrossRefGoogle Scholar
  26. Heuff RF, Swift JL, Cramb DT (2007) Fluorescence correlation spectroscopy using quantum dots: advances, challenges and opportunities. Phys Chem Chem Phys 9:1870–1880PubMedCrossRefGoogle Scholar
  27. Heuvelman G (2008) Development and design of a spatially and temporally resolved fluorescence fluctuation microscope for the analysis of molecular mobilities and interactions. PhD Thesis, Ruprecht-Karls-Universität Heidelberg, HeidelbergGoogle Scholar
  28. Hwang LC, Wohland T (2007) Recent advances in fluorescence cross-correlation spectroscopy. Cell Biochem Biophys 49:1–13PubMedCrossRefGoogle Scholar
  29. Jegou T, Chung I, Heuvelmann G, Wachsmuth M, Görisch SM, Greulich-Bode K, Boukamp P, Lichter P, Rippe K (2009) Dynamics of telomeres and promyelocytic leukemia nuclear bodies in a telomerase negative human cell line. Mol Biol Cell 20:2070–2082PubMedCrossRefGoogle Scholar
  30. Kannan B, Guo L, Sudhaharan T, Ahmed S, Maruyama I, Wohland T (2007) Spatially resolved total internal reflection fluorescence correlation microscopy using an electron multiplying charge-coupled device camera. Anal Chem 79:4463–4470PubMedCrossRefGoogle Scholar
  31. Kolin DL, Wiseman PW (2007) Advances in image correlation spectroscopy: measuring number densities, aggregation states, and dynamics of fluorescently labeled macromolecules in cells. Cell Biochem Biophys 49:141–164PubMedCrossRefGoogle Scholar
  32. Kudryavtsev V, Felekyan S, Wozniak AK, Konig M, Sandhagen C, Kuhnemuth R, Seidel CA, Oesterhelt F (2007) Monitoring dynamic systems with multiparameter fluorescence imaging. Anal Bioanal Chem 387:71–82PubMedCrossRefGoogle Scholar
  33. Lamond AI, Sleeman JE (2003) Nuclear substructure and dynamics. Curr Biol 13:R825–R828PubMedCrossRefGoogle Scholar
  34. Längst G, Becker PB (2001) Nucleosome mobilization and positioning by ISWI-containing chromatin-remodeling factors. J Cell Sci 114:2561–2568PubMedGoogle Scholar
  35. LeCaptain DJ, Van Orden A (2002) Two-beam fluorescence cross-correlation spectroscopy in an electrophoretic mobility shift assay. Anal Chem 74:1171–1176PubMedCrossRefGoogle Scholar
  36. Lumma D, Best A, Gansen A, Feuillebois F, Radler JO, Vinogradova OI (2003) Flow profile near a wall measured by double-focus fluorescence cross-correlation. Phys Rev E Stat Nonlin Soft Matter Phys 67:056313PubMedGoogle Scholar
  37. 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
  38. Magde D, Elson EL, Webb WW (1974) Fluorescence correlation spectroscopy. II: An experimental realization. Biopolymers 13:29–61PubMedCrossRefGoogle Scholar
  39. Pack C, Saito K, Tamura M, Kinjo M (2006) Microenvironment and effect of energy depletion in the nucleus analyzed by mobility of multiple oligomeric EGFPs. Biophys J 91:3921–3936PubMedCrossRefGoogle Scholar
  40. Palmer AG, Thompson NL (1987) Theory of sample translation in fluorescence correlation spectroscopy. Biophys J 51:339–343PubMedCrossRefGoogle Scholar
  41. Pan X, Foo W, Lim W, Fok MH, Liu P, Yu H, Maruyama I, Wohland T (2007) Multifunctional fluorescence correlation microscope for intracellular and microfluidic measurements. Rev Sci Instrum 78:053711PubMedCrossRefGoogle Scholar
  42. Pawley JB (ed) (1995) Handbook of biological confocal microscopy, 2nd ed. Plenum, New YorkGoogle Scholar
  43. Peters R, Peters J, Tews KH, Bahr W (1974) A microfluorimetric study of translational diffusion in erythrocyte membranes. Biochim Biophys Acta 367:282–294PubMedCrossRefGoogle Scholar
  44. Peters R, Brünger A, Schulten K (1981) Continuous fluorescence microphotolysis: a sensitive method for study of diffusion processes in single cells. Proc Natl Acad Sci USA 78:962–966PubMedCrossRefGoogle Scholar
  45. Petrasek Z, Schwille P (2008) Precise measurement of diffusion coefficients using scanning fluorescence correlation spectroscopy. Biophys J 94:1437–1448PubMedCrossRefGoogle Scholar
  46. Qian H, Elson EL (1991) Analysis of confocal laser-microscope optics for 3-D fluorescence correlation spectroscopy. Appl Opt 30:1185–1195CrossRefGoogle Scholar
  47. Rippe K, Schrader A, Riede P, Strohner R, Lehmann E, Langst G (2007) DNA sequence- and conformation-directed positioning of nucleosomes by chromatin-remodeling complexes. Proc Natl Acad Sci USA 104:15635–15640PubMedCrossRefGoogle Scholar
  48. Ruan Q, Cheng MA, Levi M, Gratton E, Mantulin WW (2004) Spatial-temporal studies of membrane dynamics: scanning fluorescence correlation spectroscopy (SFCS). Biophys J 87:1260–1267PubMedCrossRefGoogle Scholar
  49. Schwille P (2003) TIR-FCS: staying on the surface can sometimes be better. Biophys J 85:2783–2784PubMedCrossRefGoogle Scholar
  50. Sisan DR, Arevalo R, Graves C, McAllister R, Urbach JS (2006) Spatially resolved fluorescence correlation spectroscopy using a spinning disk confocal microscope. Biophys J 91:4241–4252PubMedCrossRefGoogle Scholar
  51. Skinner JP, Chen Y, Muller JD (2005) Position-sensitive scanning fluorescence correlation spectroscopy. Biophys J 89:1288–1301PubMedCrossRefGoogle Scholar
  52. van Holde KE (1989) Chromatin. Springer, HeidelbergGoogle Scholar
  53. Wachsmuth M, Weisshart K (2007) Fluorescence photobleaching and fluorescence correlation spectroscopy: two complementary technologies to study molecular dynamics in living cells. In: Shorte SL, Frischknecht F (eds) Imaging cellular and molecular biological functions. Springer, HeidelbergGoogle Scholar
  54. 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 298:677–689PubMedCrossRefGoogle Scholar
  55. Wachsmuth M, Weidemann T, Muller G, Hoffmann-Rohrer UW, Knoch TA, Waldeck W, Langowski J (2003) Analyzing intracellular binding and diffusion with continuous fluorescence photobleaching. Biophys J 84:3353–3363PubMedCrossRefGoogle Scholar
  56. Wachsmuth M, Caudron-Herger M, Rippe K (2008) Genome organization: balancing stability and plasticity. Biochim Biophys Acta 1783:2061–2079PubMedCrossRefGoogle Scholar

Copyright information

© European Biophysical Societies' Association 2009

Authors and Affiliations

  1. 1.Research Group Genome Organization and FunctionDeutsches Krebsforschungszentrum and BioQuantHeidelbergGermany
  2. 2.Cell Biology and Biophysics UnitEuropean Molecular Biology LaboratoryHeidelbergGermany

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