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Fluorescence Photobleaching and Fluorescence Correlation Spectroscopy: Two Complementary Technologies To Study Molecular Dynamics in Living Cells

  • Malte Wachsmuth
  • Klaus Weisshart
Part of the Principles and Practice book series (PRINCIPLES)

Fluorescence recovery or redistribution after photobleaching (FRAP) and fluorescence correlation or fluctuation spectroscopy (FCS) are probably the most widely used techniques employed to study the transport and diffusion as well as the interaction and immobilisation of biological molecules inside the cellular environment. This has been promoted by the emergence of fluorescent proteins for in vivo labelling and the development of confocal laser scanning microscopes that also allow for photobleaching and fluctuation spectroscopy experiments. FRAP represents a family of methods which are all based on the photoinduced bleaching (or activation) of marker molecules in selected areas of a cell followed by the relaxation back to equilibrium. FCS stands for another and complementary set of relaxation methods which are based on the observation and analysis of thermal fluctuations of sparse labelled molecules in a microscopic observation volume. Being conceptually different, these techniques taken together and combined with confocal imaging give access to a wide time and concentration range and can yield qualitatively and quantitatively biochemical and biophysical data such as concentrations, reaction rates, free and bound fractions, or diffusion coefficients. We present aspects of the biological and physical background, outline typical fields of applications, and give some guidance on how to carry out FRAP, FCS, and continuous photobleaching experiments with an emphasis on practical aspects and pitfalls.

Keywords

Green Fluorescent Protein Fluorescence Correlation Spectroscopy Confocal Volume Pinhole Size Anomalous Subdiffusion 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. Axelrod D, Koppel DE, Schlessinger J, Elson E, Webb WW (1976) Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J 16:1055–1069.PubMedGoogle Scholar
  2. Bacia K, Schwille P (2003) A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy. Methods 29:74–85.PubMedGoogle Scholar
  3. Berk DA, Yuan F, Leunig M, Jain RK (1993) Fluorescence photobleaching with spatial Fourier analysis: measurement of diffusion in light-scattering media. Biophys J 65:2428–2436.PubMedGoogle Scholar
  4. Braga J, Desterro JM, Carmo-Fonseca M (2004) Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes. Mol Biol Cell 15:4749–4760.PubMedGoogle Scholar
  5. Briddon SJ, Middleton RJ, Cordeaux Y, Flavin FM, Weinstein JA, George MW, Kellam B, Hill SJ (2004) Quantitative analysis of the formation and diffusion of A1-adenosine receptor-antagonist complexes in single living cells. Proc Natl Acad Sci USA 101:4673–4678.PubMedGoogle Scholar
  6. Brock R, Hink MA, Jovin TM (1998) Fluorescence correlation microscopy of cells in the presence of autofluorescence. Biophys J 75:2547–2557.PubMedGoogle Scholar
  7. Brown EB, Wu ES, Zipfel W, Webb WW (1999) Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery. Biophys J 77:2837–2849.PubMedGoogle Scholar
  8. Brünger A, Schulten K, Peters R (1985) Continuous fluorescence microphotolysis to observe lateral diffusion in membranes. Theoretical methods and applications. J Chem Phys 82:2147–2160.Google Scholar
  9. Calapez A, Pereira HM, Calado A, Braga J, Rino J, Carvalho C, Tavanez JP, Wahle E, Rosa AC, Carmo-Fonseca M (2002) The intranuclear mobility of messenger RNA binding proteins is ATP dependent and temperature sensitive. J Cell Biol 159:795–805.PubMedGoogle Scholar
  10. Campbell RE, Tour O, Palmer AE, Steinbach PA, Baird GS, Zacharias DA, Tsien RY (2002) A monomeric red fluorescent protein. Proc Natl Acad Sci USA 99:7877–7882.PubMedGoogle Scholar
  11. Carmo-Fonseca M, Platani M, Swedlow JR (2002) Macromolecular mobility inside the cell nucleus. Trends Cell Biol 12:491–495.PubMedGoogle Scholar
  12. Carrero G, McDonald D, Crawford E, de Vries G, Hendzel MJ (2003) Using FRAP and mathematical modeling to determine the in vivo kinetics of nuclear proteins. Methods 29:14–28.PubMedGoogle Scholar
  13. Carrero G, Crawford E, Th’ng J, de Vries G, Hendzel MJ (2004) Quantification of protein-protein and protein-DNA interactions in vivo, using fluorescence recovery after photobleaching. Methods Enzymol 375:415–442.PubMedGoogle Scholar
  14. Chen Y, Muller JD, So PT, Gratton E (1999) The photon counting histogram in fluorescence fluctuation spectroscopy. Biophys J 77:553–567.PubMedGoogle Scholar
  15. 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 273:797–801.PubMedGoogle Scholar
  16. 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 180:131–139.Google Scholar
  17. Digman MA, Brown CM, Sengupta P, Wiseman PW, Horwitz AR, Gratton E (2005) Measuring fast dynamics in solutions and cells with a laser scanning microscope. Biophys J 89:1317–1327.PubMedGoogle Scholar
  18. Doose S, Tsay JM, Pinaud F, Weiss S (2005) Comparison of photophysical and colloidal properties of biocompatible semiconductor nanocrystals using fluorescence correlation spectroscopy. Anal Chem 77:2235–2242.PubMedGoogle Scholar
  19. Dundr M, Hoffmann-Rohrer U, Hu Q, Grummt I, Rothblum LI, Phair RD, Misteli T (2002) A kinetic framework for a mammalian RNA polymerase in vivo. Science 298:1623–1626.PubMedGoogle Scholar
  20. Edidin M, Zagyansky Y, Lardner TJ (1976) Measurement of membrane protein lateral diffusion in single cells. Science 191:466–468.PubMedGoogle Scholar
  21. Ellenberg J, Siggia ED, Moreira JE, Smith CL, Presley JF, Worman HJ, Lippincott-Schwartz J (1997) Nuclear membrane dynamics and reassembly in living cells: targeting of an inner nuclear membrane protein in interphase and mitosis. J Cell Biol 138:1193–1206.PubMedGoogle Scholar
  22. Elsner M, Hashimoto H, Simpson JC, Cassel D, Nilsson T, Weiss M (2003) Spatiotemporal dynamics of the COPI vesicle machinery. EMBO Rep 4:1000–1004.PubMedGoogle Scholar
  23. Elson EL (2004) Quick tour of fluorescence correlation spectroscopy from its inception. J Biomed Opt 9:857–864.PubMedGoogle Scholar
  24. Elson EL, Magde D (1974) Fluorescence correlation spectroscopy. I. Conceptual basis and theory. Biopolymers 13:1–27.Google Scholar
  25. Fradin C, Abu-Arish A, Granek R, Elbaum M (2003) Fluorescence correlation spectroscopy close to a fluctuating membrane. Biophys J 84:2005–2020.PubMedGoogle Scholar
  26. Gennerich A, Schild D (2000) Fluorescence correlation spectroscopy in small cytosolic compartments depends critically on the diffusion model used. Biophys J 79:3294–3306.PubMedGoogle Scholar
  27. 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 101:13221–13226.PubMedGoogle Scholar
  28. Gösch M, Rigler R (2005) Fluorescence correlation spectroscopy of molecular motions and kinetics. Adv Drug Deliv Rev 57:169–190.PubMedGoogle Scholar
  29. Griffin BA, Adams SR, Jones J, Tsien RY (2000) Fluorescent labeling of recombinant proteins in living cells with FlAsH. Methods Enzymol 327:565–578.PubMedGoogle Scholar
  30. Grünwald D, Cardoso MC, Leonhardt H, Buschmann V (2005) Diffusion and binding properties investigated by fluorescence correlation spectroscopy (FCS). Curr Pharm Biotechnol 6:381–386.PubMedGoogle Scholar
  31. Harms GS, Cognet L, Lommerse PH, Blab GA, Schmidt T (2001) Autofluorescent proteins in single-molecule research: applications to live cell imaging microscopy. Biophys J 80:2396–2408.PubMedGoogle Scholar
  32. 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 95:13573–13578.PubMedGoogle Scholar
  33. Haustein E, Schwille P (2003) Ultrasensitive investigations of biological systems by fluorescence correlation spectroscopy. Methods 29:153–166.PubMedGoogle Scholar
  34. Hecht B (2004) Nano-optics with single quantum systems. Philos Trans R Soc Lond Ser A 362:881–899.Google Scholar
  35. Helmchen F, Denk W (2005) Deep tissue two-photon microscopy. Nat Methods 2:932–940.PubMedGoogle Scholar
  36. Hess ST, Huang S, Heikal AA, Webb WW (2002) Biological and chemical applications of fluorescence correlation spectroscopy: a review. Biochemistry 41:697–705.PubMedGoogle Scholar
  37. Hink MA, Bisselin T, Visser AJ (2002) Imaging protein-protein interactions in living cells. Plant Mol Biol 50:871–883.PubMedGoogle Scholar
  38. Hopt A, Neher E (2001) Highly nonlinear photodamage in two-photon fluorescence microscopy. Biophys J 80:2029–2036.PubMedGoogle Scholar
  39. Houtsmuller AB, Vermeulen W (2001) Macromolecular dynamics in living cell nuclei revealed by fluorescence redistribution after photobleaching. Histochem Cell Biol 115:13–21.PubMedGoogle Scholar
  40. Houtsmuller AB, Rademakers S, Nigg AL, Hoogstraten D, Hoeijmakers JH, Vermeulen W (1999) Action of DNA repair endonuclease ERCC1/XPF in living cells. Science 284:958–961.PubMedGoogle Scholar
  41. Hwang LC, Wohland T (2005) Single wavelength excitation fluorescence cross-correlation spectroscopy with spectrally similar fluorophores: resolution for binding studies. J Chem Phys 122:114708.PubMedGoogle Scholar
  42. Jahnz M, Schwille P (2004) Enzyme assays for confocal single molecule spectroscopy. Curr Pharm Biotechnol 5:221–229.PubMedGoogle Scholar
  43. Jankevics H, Prummer M, Izewska P, Pick H, Leufgen K, Vogel H (2005) Diffusion-time distribution analysis reveals characteristic ligand-dependent interaction patterns of nuclear receptors in living cells. Biochemistry 44:11676–11683.PubMedGoogle Scholar
  44. 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 96:13756–13761.PubMedGoogle Scholar
  45. Kimura H, Cook PR (2001) Kinetics of core histones in living human cells: little exchange of H3 and H4 and some rapid exchange of H2B. J Cell Biol 153:1341–1353.PubMedGoogle Scholar
  46. Kimura H, Hieda M, Cook PR (2004) Measuring histone and polymerase dynamics in living cells. Methods Enzymol 375:381–393.PubMedGoogle Scholar
  47. Klonis N, Rug M, Harper I, Wickham M, Cowman A, Tilley L (2002) Fluorescence photobleaching analysis for the study of cellular dynamics. Eur Biophys J 31:36–51.PubMedGoogle Scholar
  48. Kohl T, Schwille P (2005) Fluorescence correlation spectroscopy with autofluorescent proteins. Adv Biochem Eng Biotechnol 95:107–142.PubMedGoogle Scholar
  49. Kubitscheck U, Wedekind P, Peters R (1994) Lateral diffusion measurement at high spatial resolution by scanning microphotolysis in a confocal microscope. Biophys J 67:948–956.PubMedGoogle Scholar
  50. Kubitscheck U, Heinrich O, Peters R (1996) Continuous scanning micro-photolysis: a simple laser scanning microscopic method for lateral transport measurements employing single- or two-photon excitation. Bioimaging 4:158–167.Google Scholar
  51. Lakowicz JR (1999) Principles of fluorescence spectroscopy. Kluwer/Plenum, New York.Google Scholar
  52. Levi V, Ruan Q, Kis-Petikova K, Gratton E (2003) Scanning FCS, a novel method for three-dimensional particle tracking. Biochem Soc Trans 31:997–1000.PubMedGoogle Scholar
  53. Lippincott-Schwartz J, Snapp E, Kenworthy A (2001) Studying protein dynamics in living cells. Nat Rev Mol Cell Biol 2:444–456.PubMedGoogle Scholar
  54. Lumma D, Keller S, Vilgis T, Radler JO (2003) Dynamics of large semiflexible chains probed by fluorescence correlation spectroscopy. Phys Rev Lett 90:218301.PubMedGoogle Scholar
  55. Magde D, Elson EL, Webb WW (1972) Thermodynamic fluctuations in a reacting system–measurement by fluorescence correlation spectroscopy. Phys Rev Lett 29:705–708.Google Scholar
  56. Magde D, Elson EL, Webb WW (1974) Fluorescence correlation spectroscopy. II. An experimental realization. Biopolymers 13:29–61.PubMedGoogle Scholar
  57. McGrath JL, Tardy Y, Dewey CF Jr, Meister JJ, Hartwig JH (1998) Simultaneous measurements of actin filament turnover, filament fraction, and monomer diffusion in endothelial cells. Biophys J 75:2070–2078.PubMedGoogle Scholar
  58. Medina MA, Schwille P (2002) Fluorescence correlation spectroscopy for the detection and study of single molecules in biology. BioEssays 24:758–764.PubMedGoogle Scholar
  59. Medintz IL, Uyeda HT, Goldman ER, Mattoussi H (2005) Quantum dot bioconjugates for imaging, labelling and sensing. Nat Mater 4:435–446.PubMedGoogle Scholar
  60. Meseth U, Wohland T, Rigler R, Vogel H (1999) Resolution of fluorescence correlation measurements. Biophys J 76:1619–1631.PubMedGoogle Scholar
  61. Michalet X, Pinaud FF, Bentolila LA, Tsay JM, Doose S, Li JJ, Sundaresan G, Wu AM, Gambhir SS, Weiss S (2005) Quantum dots for live cells, in vivo imaging, and diagnostics. Science 307:538–544.PubMedGoogle Scholar
  62. Misteli T (2001) Protein dynamics: Implications for nuclear architecture and gene expression. Science 291:843–847.PubMedGoogle Scholar
  63. Modos K, Galantai R, Bardos-Nagy I, Wachsmuth M, Toth K, Fidy J, Langowski J (2004) Maximum-entropy decomposition of fluorescence correlation spectroscopy data: application to liposome-human serum albumin association. Eur Biophys J 33:59–67.PubMedGoogle Scholar
  64. Muller JD, Gratton E (2003) High-pressure fluorescence correlation spectroscopy. Biophys J 85:2711–2719.PubMedGoogle Scholar
  65. Nirmal M, Dabbousi BO, Bawendi MG, Macklin JJ, Trautman JK, Harris TD, Brus LE (1996) Fluorescence intermittency in single cadmium selenide nanocrystals. Nature 383:802–804.Google Scholar
  66. Oancea E, Teruel MN, Quest AF, Meyer T (1998) Green fluorescent protein (GFP)-tagged cysteine-rich domains from protein kinase C as fluorescent indicators for diacylglycerol signaling in living cells. J Cell Biol 140:485–498.PubMedGoogle Scholar
  67. Patterson GH, Knobel SM, Sharif WD, Kain SR, Piston DW (1997) Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys J 73:2782–2790.PubMedGoogle Scholar
  68. Patterson GH, Lippincott-Schwartz J (2002) A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297:1873–1877.PubMedGoogle Scholar
  69. Pawley JB (ed) (1995) Handbook of biological confocal microscopy. Plenum, New York.Google Scholar
  70. Periasamy N, Verkman AS (1998) Analysis of fluorophore diffusion by continuous distributions of diffusion coefficients: application to photobleaching measurements of multicomponent and anomalous diffusion. Biophys J 75:557–567.PubMedGoogle Scholar
  71. Periasamy N, Bicknese S, Verkman AS (1996) Reversible photobleaching of fluorescein conjugates in air-saturated viscous solutions: singlet and triplet state quenching by tryptophan. Photochem Photobiol 63:265–271.PubMedGoogle Scholar
  72. Peters R (1983) Fluorescence microphotolysis. diffusion measurements in single cells. Naturwissenschaften 70:294–302.PubMedGoogle Scholar
  73. Peters R, Kubitscheck U (1999) Scanning microphotolysis: Three-dimensional diffusion measurement and optical single-transporter recording. Methods 18:508–517.PubMedGoogle Scholar
  74. Peters R, Peters J, Tews KH, Bahr W (1974) A microfluorimetric study of translational diffusion in erythrocyte membranes. Biochim Biophys Acta 367:282–294.PubMedGoogle Scholar
  75. 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 78:962–966.PubMedGoogle Scholar
  76. Phair RD, Misteli T (2001) Kinetic modelling approaches to in vivo imaging. Nat Rev Mol Cell Biol 2:898–907.PubMedGoogle Scholar
  77. Phair RD, Gorski SA, Misteli T (2004a) Measurement of dynamic protein binding to chromatin in vivo, using photobleaching microscopy. Methods Enzymol 375:393–414.PubMedGoogle Scholar
  78. Phair RD, Scaffidi P, Elbi C, Vecerova J, Dey A, Ozato K, Brown DT, Hager G, Bustin M, Misteli T (2004b) Global nature of dynamic protein-chromatin interactions in vivo: three-dimensional genome scanning and dynamic interaction networks of chromatin proteins. Mol Cell Biol 24:6393–6402.PubMedGoogle Scholar
  79. Politz JC (1999) Use of caged fluorochromes to track macromolecular movement in living cells. Trends Cell Biol 9:284–287.PubMedGoogle Scholar
  80. Potma EO, Boeij WP, Bosgraaf L, Roelofs J, van Haastert PJ, Wiersma DA (2001) Reduced protein diffusion rate by cytoskeleton in vegetative and polarized dictyostelium cells. Biophys J 81:2010–2019.PubMedGoogle Scholar
  81. Rabut G, Ellenberg J (2005) Photobleaching techniques to study mobility and molecular dynamics of proteins in live cells: FRAP, iFRAP, and FLIP. In: Spector D, Goldman D (eds) Live cell imaging: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 101–126.Google Scholar
  82. Reits EA, Neefjes JJ (2001) From fixed to frap: measuring protein mobility and activity in living cells. Nat Cell Biol 3:E145–147.PubMedGoogle Scholar
  83. Ricka J, Binkert T (1989) Direct measurement of a distinct correlation function by fluorescence cross correlation. Phys Rev A 39:2646–2652.Google Scholar
  84. Rigler R, Elson ES (eds) (2001) Fluorescence correlation spectroscopy: theory and applications. Springer, Berlin.Google Scholar
  85. Rigler R, Mets Ü, Widengren J, Kask P (1993) Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion. Eur Biophys J 22:169–1-75.Google Scholar
  86. Rizzo MA, Springer GH, Granada B, Piston DW (2004) An improved cyan fluorescent protein variant useful for FRET. Nat Biotechnol 22:445–449.PubMedGoogle Scholar
  87. Sanchez SA, Gratton E (2005) Lipid-protein interactions revealed by two-photon microscopy and fluorescence correlation spectroscopy. Acc Chem Res 38:469–477.PubMedGoogle Scholar
  88. Saxton MJ (2001) Anomalous subdiffusion in fluorescence photobleaching recovery: a Monte Carlo study. Biophys J 81:2226–2240.PubMedGoogle Scholar
  89. Saxton MJ, Jacobson K (1997) Single-particle tracking: applications to membrane dynamics. Annu Rev Biophys Biomol Struct 26:373–399.PubMedGoogle Scholar
  90. Schulten K (1986) Continuous fluorescence microphotolysis by a sin2 kx grating. Chem Phys Lett 124:230–236.Google Scholar
  91. Schwille P (2001) Fluorescence correlation spectroscopy and its potential for intracellular applications. Cell Biochem Biophys 34:383–408.PubMedGoogle Scholar
  92. Schwille P, Meyer-Almes FJ, Rigler R (1997) Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution. Biophys J 72:1878–1886.PubMedGoogle Scholar
  93. Schwille P, Korlach J, Webb WW (1999) Fluorescence correlation spectroscopy with single-molecule sensitivity on cell and model membranes. Cytometry 36:176–182.PubMedGoogle Scholar
  94. 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. Proc Natl Acad Sci USA 97:151–156.PubMedGoogle Scholar
  95. Sengupta P, Garai K, Balaji J, Periasamy N, Maiti S (2003) Measuring size distribution in highly heterogeneous systems with fluorescence correlation spectroscopy. Biophys J 84:1977–1984.PubMedGoogle Scholar
  96. Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909.PubMedGoogle Scholar
  97. Siggia ED, Lippincott-Schwartz J, Bekiranov S (2000) Diffusion in inhomogeneous media: theory and simulations applied to whole cell photobleach recovery. Biophys J 79:1761–1770.PubMedGoogle Scholar
  98. Skakun VV, Hink MA, Digris AV, Engel R, Novikov EG, Apanasovich VV, Visser AJ (2005) Global analysis of fluorescence fluctuation data. Eur Biophys J 34:323–334.PubMedGoogle Scholar
  99. Song L, Hennink EJ, Young IT, Tanke HJ (1995) Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy. Biophys J 68:2588–2600.PubMedGoogle Scholar
  100. Song L, Varma CA, Verhoeven JW, Tanke HJ (1996) Influence of the triplet excited state on the photobleaching kinetics of fluorescein in microscopy. Biophys J 70:2959–2968.PubMedGoogle Scholar
  101. Song L, van Gijlswijk RP, Young IT, Tanke HJ (1997) Influence of fluorochrome labeling density on the photobleaching kinetics of fluorescein in microscopy. Cytometry 27:213–223.PubMedGoogle Scholar
  102. Soumpasis DM (1983) Theoretical analysis of fluorescence photobleaching recovery experiments. Biophys J 41:95–97.PubMedGoogle Scholar
  103. Sprague BL, Pego RL, Stavreva DA, McNally JG (2004) Analysis of binding reactions by fluorescence recovery after photobleaching. Biophys J 86:3473–3495.PubMedGoogle Scholar
  104. Starr TE, Thompson NL (2002) Fluorescence pattern photobleaching recovery for samples with multi-component diffusion. Biophys Chem 97:29–44.PubMedGoogle Scholar
  105. Stavreva DA, McNally JG (2004) Fluorescence recovery after photobleaching (FRAP) methods for visualizing protein dynamics in living mammalian cell nuclei. Methods Enzymol 375:443–455.PubMedGoogle Scholar
  106. Stout AL, Axelrod D (1995) Spontaneous recovery of fluorescence by photobleached surface-adsorbed proteins. Photochem Photobiol 62:239–244.PubMedGoogle Scholar
  107. Strohner R, Wachsmuth M, Dachauer K, Mazurkiewicz J, Hochstätter J, Rippe K, Längst G (2005) A ‘loop recapture’ mechanism for ACF-dependent nucleosome remodeling. Nat Struct Mol Biol 12:683–690.PubMedGoogle Scholar
  108. Subramanian K, Meyer T (1997) Calcium-induced restructuring of nuclear envelope and endoplasmic reticulum calcium stores. Cell 89:963–971.PubMedGoogle Scholar
  109. Surrey T, Elowitz MB, Wolf PE, Yang F, Nedelec F, Shokat K, Leibler S (1998) Chromophore-assisted light inactivation and self-organization of microtubules and motors. Proc Natl Acad Sci USA 95:4293–4298.PubMedGoogle Scholar
  110. Svoboda K, Yasuda R (2006) Principles of two-photon excitation microscopy and its applications to neuroscience. Neuron 50:823–839.PubMedGoogle Scholar
  111. Swaminathan R, Hoang CP, Verkman AS (1997) Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion. Biophys J 72:1900–1907.PubMedGoogle Scholar
  112. Tardy Y, McGrath JL, Hartwig JH, Dewey CF (1995) Interpreting photoactivated fluorescence microscopy measurements of steady-state actin dynamics. Biophys J 69:1674–1682.PubMedGoogle Scholar
  113. Thompson NL (1991) Fluorescence correlation spectroscopy. In: Lakowicz JR (ed) Topics in fluorescence spectroscopy, vol 1. Plenum, New York, pp 337–378.Google Scholar
  114. Thompson NL, Lieto AM, Allen NW (2002) Recent advances in fluorescence correlation spectroscopy. Curr Opin Struct Biol 12:634–641.PubMedGoogle Scholar
  115. Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544.PubMedGoogle Scholar
  116. Umenishi F, Verbavatz JM, Verkman AS (2000) cAMP regulated membrane diffusion of a green fluorescent protein-aquaporin 2 chimera. Biophys J 78:1024–1035.PubMedGoogle Scholar
  117. Van Keuren E, Schrof W (2003) Fluorescence recovery after two-photon bleaching for the study of dye diffusion in polymer systems. Macromolecules 36:5002–5007.Google Scholar
  118. Verkman AS (2002) Solute and macromolecule diffusion in cellular aqueous compartments. Trends Biochem Sci 27:27–33.PubMedGoogle Scholar
  119. Verkman AS (2003) Diffusion in cells measured by fluorescence recovery after photobleaching. Methods Enzymol 360:635–648.PubMedGoogle Scholar
  120. Vukojevic V, Pramanik A, Yakovleva T, Rigler R, Terenius L, Bakalkin G (2005) Study of molecular events in cells by fluorescence correlation spectroscopy. Cell Mol Life Sci 62:535–550.PubMedGoogle Scholar
  121. 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 298:677–689.PubMedGoogle Scholar
  122. 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 84:3353–3363.PubMedGoogle Scholar
  123. Wedekind P, Kubitscheck U, Heinrich O, Peters R (1996) Line-scanning microphotolysis for diffraction-limited measurements of lateral diffusion. Biophys J 71:1621–1632.PubMedGoogle Scholar
  124. Wedekind P, Kubitscheck U, Peters R (1994) Scanning microphotolysis: a new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging. J Microsc 176:23–33.PubMedGoogle Scholar
  125. Weidemann T, Wachsmuth M, Tewes M, Rippe K, Langowski J (2002) Analysis of ligand binding by two-colour fluorescence cross-correlation spectroscopy. Single Mol 3:49–61.Google Scholar
  126. Weidemann T, Wachsmuth M, Knoch TA, Muller G, Waldeck W, Langowski J (2003) Counting nucleosomes in living cells with a combination of fluorescence correlation spectroscopy and confocal imaging. J Mol Biol 334:229–240.PubMedGoogle Scholar
  127. Weisshart K, Jungel V, Briddon SJ (2004) The LSM 510 META–ConfoCor 2 system: an integrated imaging and spectroscopic platform for single-molecule detection. Curr Pharm Biotechnol 5:135–154.PubMedGoogle Scholar
  128. White J, Stelzer E (1999) Photobleaching GFP reveals protein dynamics inside live cells. Trends Cell Biol 9:61–65.PubMedGoogle Scholar
  129. Widengren J, Mets Ü, Rigler R (1995) Fluorescence correlation spectroscopy of triplet states in solution: a theoretical and experimental study. J Phys Chem 99:13368–13379.Google Scholar
  130. Zhang J, Campbell RE, Ting AY, Tsien RY (2002) Creating new fluorescent probes for cell biology. Nat Rev Mol Cell Biol 3:906–918.PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2007

Authors and Affiliations

  • Malte Wachsmuth
    • 1
  • Klaus Weisshart
    • 2
  1. 1.Cell Biophysics GroupInstitut Pasteur KoreaSeongbukgu, SeoulRepublic of Korea
  2. 2.Carl Zeiss MicroImaging GmbHCarl-Zeiss-Promenade 10JenaGermany

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