Abstract

The intracellular mobility of biomolecules is determined by transport and diffusion as well as molecular interactions and is crucial for many processes in living cells. Methods of fluorescence microscopy like confocal laser scanning microscopy (CLSM) can be used to characterize the intracellular distribution of fluorescently labeled biomolecules. Fluorescence correlation spectroscopy (FCS) is used to describe diffusion, transport and photo-physical processes quantitatively. As an alternative to FCS, spatially resolved measurements of mobilities can be implemented using a CLSM by utilizing the spatio-temporal information inscribed into the image by the scan process, referred to as raster image correlation spectroscopy (RICS). Here we present and discuss an extended approach, multiple scan speed image correlation spectroscopy (msICS), which benefits from the advantages of RICS, i.e. the use of widely available instrumentation and the extraction of spatially resolved mobility information, without the need of a priori knowledge of diffusion properties. In addition, msICS covers a broad dynamic range, generates correlation data comparable to FCS measurements, and allows to derive two-dimensional maps of diffusion coefficients. We show the applicability of msICS to fluorophores in solution and to free EGFP in living cells.

© 2010 OSA

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. E. L. Elson and D. Magde, “Fluorescence correlation spectroscopy. I. Conceptual basis and theory,” Biopolymers 13(1), 1–27 (1974).
    [CrossRef]
  2. D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974).
    [CrossRef] [PubMed]
  3. D. A. Bulseco, D. E. Wolf, S. Greenfield, and E. W. David, “Fluorescence Correlation Spectroscopy: Molecular Complexing in Solution and in Living Cells,” in Digital Microscopy, 3rd Edition (Academic Press, 2007), pp. 525–559.
  4. D. Grünwald, M. C. Cardoso, H. Leonhardt, and V. Buschmann, “Diffusion and binding properties investigated by Fluorescence Correlation Spectroscopy (FCS),” Curr. Pharm. Biotechnol. 6(5), 381–386 (2005).
    [CrossRef] [PubMed]
  5. M. Wachsmuth, and K. Weisshart, “Fluorescence photobleaching and fluorescence correlation spectroscopy: two complementary technologies to study molecular dynamics in living cells,” in Imaging Cellular and Molecular Biological Functions (Springer Verlag, Heidelberg, 2007).
  6. W. B. Amos and J. G. White, “How the confocal laser scanning microscope entered biological research,” Biol. Cell 95(6), 335–342 (2003).
    [CrossRef] [PubMed]
  7. C. Cremer and T. Cremer, “Considerations on a laser-scanning-microscope with high resolution and depth of field,” Microsc. Acta 81(1), 31–44 (1978).
    [PubMed]
  8. J. Pawley, Handbook of Biological Confocal Microscopy (Springer, Berlin, 2006).
  9. G. Rabut, J. Ellenberg, D. Spector, and D. Goldman, “Photobleaching Techniques to Study Mobility and Molecular Dynamics of Proteins in Live Cells: FRAP, iFRAP, and FLIP,” in Live Cell Imaging - A Laboratory Manual (CSHL Press, Cold Spring Harbor, 2005), pp. 101–126.
  10. M. E. van Royen, P. Farla, K. A. Mattern, B. Geverts, J. Trapman, and A. B. Houtsmuller, “Fluorescence recovery after photobleaching (FRAP) to study nuclear protein dynamics in living cells,” Methods Mol. Biol. 464, 363–385 (2009).
    [CrossRef]
  11. N. O. Petersen, P. L. Höddelius, P. W. Wiseman, O. Seger, and K. E. Magnusson, “Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application,” Biophys. J. 65(3), 1135–1146 (1993).
    [CrossRef] [PubMed]
  12. N. O. Petersen, R. Rigler, and E. S. Elson, “FCS and spatial correlations on biological surfaces,” in Fluorescence Correlation Spectroscopy - Theory and Applications (Springer, Heidelberg, 2001), pp. 162–184.
  13. D. L. Kolin, S. Costantino, and P. W. Wiseman, “Sampling effects, noise, and photobleaching in temporal image correlation spectroscopy,” Biophys. J. 90(2), 628–639 (2006).
    [CrossRef]
  14. D. L. Kolin, D. Ronis, and P. W. Wiseman, “k-Space image correlation spectroscopy: a method for accurate transport measurements independent of fluorophore photophysics,” Biophys. J. 91(8), 3061–3075 (2006).
    [CrossRef] [PubMed]
  15. D. L. Kolin and P. W. Wiseman, “Advances in image correlation spectroscopy: measuring number densities, aggregation states, and dynamics of fluorescently labeled macromolecules in cells,” Cell Biochem. Biophys. 49(3), 141–164 (2007).
    [CrossRef] [PubMed]
  16. C. M. Brown, R. B. Dalal, B. Hebert, M. A. Digman, A. R. Horwitz, and E. Gratton, “Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope,” J. Microsc. 229(1), 78–91 (2008).
    [CrossRef] [PubMed]
  17. M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89(2), 1317–1327 (2005).
    [CrossRef] [PubMed]
  18. M. A. Digman and E. Gratton, “Analysis of diffusion and binding in cells using the RICS approach,” Microsc. Res. Tech. 72(4), 323–332 (2009).
    [CrossRef]
  19. M. A. Digman, P. W. Wiseman, C. Choi, A. R. Horwitz, and E. Gratton, “Stoichiometry of molecular complexes at adhesions in living cells,” Proc. Natl. Acad. Sci. U.S.A. 106(7), 2170–2175 (2009).
    [CrossRef] [PubMed]
  20. E. Gielen, N. Smisdom, M. Vandeven, B. De Clercq, E. Gratton, M. Digman, J.-M. Rigo, J. Hofkens, Y. Engelborghs, and M. Ameloot, “Measuring Diffusion of Lipid-like Probes in Artificial and Natural Membranes by Raster Image Correlation Spectroscopy (RICS): Use of a Commercial Laser-Scanning Microscope with Analog Detection,” Langmuir (2009).
  21. M. A. Digman, R. Dalal, A. F. Horwitz, and E. Gratton, “Mapping the number of molecules and brightness in the laser scanning microscope,” Biophys. J. 94(6), 2320–2332 (2008).
    [CrossRef]
  22. V. Vukojević, M. Heidkamp, Y. Ming, B. Johansson, L. Terenius, and R. Rigler, “Quantitative single-molecule imaging by confocal laser scanning microscopy,” Proc. Natl. Acad. Sci. U.S.A. 105(47), 18176–18181 (2008).
    [CrossRef] [PubMed]
  23. K. M. Berland, P. T. C. So, Y. Chen, W. W. Mantulin, and E. Gratton, “Scanning two-photon fluctuation correlation spectroscopy: particle counting measurements for detection of molecular aggregation,” Biophys. J. 71(1), 410–420 (1996).
    [CrossRef] [PubMed]
  24. D. E. Koppel, F. Morgan, A. E. Cowan, and J. H. Carson, “Scanning concentration correlation spectroscopy using the confocal laser microscope,” Biophys. J. 66(2), 502–507 (1994).
    [CrossRef] [PubMed]
  25. J. Ries, S. Chiantia, and P. Schwille, “Accurate determination of membrane dynamics with line-scan FCS,” Biophys. J. 96(5), 1999–2008 (2009).
    [CrossRef] [PubMed]
  26. J. Widengren, Ü. Mets, and R. Rigler, “Fluorescence Correlation Spectroscopy of Triplet States in Solution: A Theoretical and Experimental Study,” J. Phys. Chem. 99(36), 13368–13379 (1995).
    [CrossRef]
  27. “LFD Workshop 2006 - Laboratory for Fluorescence Dynamics” (2006), http://www.lfd.uci.edu/workshop/2006/ .
  28. M. Wachsmuth, “Method for measuring fluorescence fluctuations in the presence of slow signal fluctuations,” US Patent No. 7,154,602 (2006).
  29. G. S. Harms, L. Cognet, P. H. Lommerse, G. A. Blab, and T. Schmidt, “Autofluorescent proteins in single-molecule research: applications to live cell imaging microscopy,” Biophys. J. 80, 2396–2408 (2001).
    [CrossRef] [PubMed]
  30. J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Kluwer Academic/Plenum Publishers, New York, 1999).
  31. M. Wachsmuth, W. Waldeck, and J. Langowski, “Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy,” J. Mol. Biol. 298(4), 677–689 (2000).
    [CrossRef] [PubMed]
  32. I. Gregor, D. Patra, and J. Enderlein, “Optical saturation in fluorescence correlation spectroscopy under continuous-wave and pulsed excitation,” ChemPhysChem 6(1), 164–170 (2005).
    [CrossRef] [PubMed]
  33. P. Dittrich, F. Malvezzi-Campeggi, M. Jahnz, and P. Schwille, “Accessing molecular dynamics in cells by fluorescence correlation spectroscopy,” Biol. Chem. 382(3), 491–494 (2001).
    [CrossRef] [PubMed]
  34. P. Schwille, U. Haupts, S. Maiti, and W. W. Webb, “Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation,” Biophys. J. 77(4), 2251–2265 (1999).
    [CrossRef] [PubMed]
  35. K. P. Müller, F. Erdel, M. Caudron-Herger, C. Marth, B. D. Fodor, M. Richter, M. Scaranaro, J. Beaudouin, M. Wachsmuth, and K. Rippe, “Multiscale analysis of dynamics and interactions of heterochromatin protein 1 by fluorescence fluctuation microscopy,” Biophys. J. 97(11), 2876–2885 (2009).
    [CrossRef] [PubMed]
  36. J. Beaudouin, F. Mora-Bermúdez, T. Klee, N. Daigle, and J. Ellenberg, “Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins,” Biophys. J. 90(6), 1878–1894 (2006).
    [CrossRef] [PubMed]
  37. C. Pack, K. Saito, M. Tamura, and M. Kinjo, “Microenvironment and effect of energy depletion in the nucleus analyzed by mobility of multiple oligomeric EGFPs,” Biophys. J. 91(10), 3921–3936 (2006).
    [CrossRef] [PubMed]
  38. M. Wachsmuth, M. Caudron-Herger, and K. Rippe, “Genome organization: balancing stability and plasticity,” Biochim. Biophys. Acta 1783(11), 2061–2079 (2008).
    [CrossRef] [PubMed]
  39. N. Dross, C. Spriet, M. Zwerger, G. Müller, W. Waldeck, J. Langowski, and J. Z. Rappoport, “Mapping eGFP oligomer mobility in living cell nuclei,” PLoS ONE 4(4), e5041–e5041 (2009).
    [CrossRef] [PubMed]
  40. A. Bancaud, S. Huet, N. Daigle, J. Mozziconacci, J. Beaudouin, and J. Ellenberg, “Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin,” EMBO J. 28(24), 3785–3798 (2009).
    [CrossRef] [PubMed]
  41. C. M. Roth, P. I. Heinlein, M. Heilemann, and D.-P. Herten, “Imaging diffusion in living cells using time-correlated single-photon counting,” Anal. Chem. 79(19), 7340–7345 (2007).
    [CrossRef] [PubMed]
  42. G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
    [CrossRef] [PubMed]
  43. B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially Resolved Total Internal Reflection Fluorescence Correlation Microscopy Using an Electron Multiplying Charge-Coupled Device Camera,” Analytical Chemistry (2007).
  44. D. J. Needleman, Y. Xu, and T. J. Mitchison, “Pin-hole array correlation imaging: highly parallel fluorescence correlation spectroscopy,” Biophys. J. 96(12), 5050–5059 (2009).
    [CrossRef] [PubMed]

2009 (9)

M. E. van Royen, P. Farla, K. A. Mattern, B. Geverts, J. Trapman, and A. B. Houtsmuller, “Fluorescence recovery after photobleaching (FRAP) to study nuclear protein dynamics in living cells,” Methods Mol. Biol. 464, 363–385 (2009).
[CrossRef]

M. A. Digman and E. Gratton, “Analysis of diffusion and binding in cells using the RICS approach,” Microsc. Res. Tech. 72(4), 323–332 (2009).
[CrossRef]

M. A. Digman, P. W. Wiseman, C. Choi, A. R. Horwitz, and E. Gratton, “Stoichiometry of molecular complexes at adhesions in living cells,” Proc. Natl. Acad. Sci. U.S.A. 106(7), 2170–2175 (2009).
[CrossRef] [PubMed]

J. Ries, S. Chiantia, and P. Schwille, “Accurate determination of membrane dynamics with line-scan FCS,” Biophys. J. 96(5), 1999–2008 (2009).
[CrossRef] [PubMed]

K. P. Müller, F. Erdel, M. Caudron-Herger, C. Marth, B. D. Fodor, M. Richter, M. Scaranaro, J. Beaudouin, M. Wachsmuth, and K. Rippe, “Multiscale analysis of dynamics and interactions of heterochromatin protein 1 by fluorescence fluctuation microscopy,” Biophys. J. 97(11), 2876–2885 (2009).
[CrossRef] [PubMed]

N. Dross, C. Spriet, M. Zwerger, G. Müller, W. Waldeck, J. Langowski, and J. Z. Rappoport, “Mapping eGFP oligomer mobility in living cell nuclei,” PLoS ONE 4(4), e5041–e5041 (2009).
[CrossRef] [PubMed]

A. Bancaud, S. Huet, N. Daigle, J. Mozziconacci, J. Beaudouin, and J. Ellenberg, “Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin,” EMBO J. 28(24), 3785–3798 (2009).
[CrossRef] [PubMed]

G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
[CrossRef] [PubMed]

D. J. Needleman, Y. Xu, and T. J. Mitchison, “Pin-hole array correlation imaging: highly parallel fluorescence correlation spectroscopy,” Biophys. J. 96(12), 5050–5059 (2009).
[CrossRef] [PubMed]

2008 (4)

M. Wachsmuth, M. Caudron-Herger, and K. Rippe, “Genome organization: balancing stability and plasticity,” Biochim. Biophys. Acta 1783(11), 2061–2079 (2008).
[CrossRef] [PubMed]

C. M. Brown, R. B. Dalal, B. Hebert, M. A. Digman, A. R. Horwitz, and E. Gratton, “Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope,” J. Microsc. 229(1), 78–91 (2008).
[CrossRef] [PubMed]

M. A. Digman, R. Dalal, A. F. Horwitz, and E. Gratton, “Mapping the number of molecules and brightness in the laser scanning microscope,” Biophys. J. 94(6), 2320–2332 (2008).
[CrossRef]

V. Vukojević, M. Heidkamp, Y. Ming, B. Johansson, L. Terenius, and R. Rigler, “Quantitative single-molecule imaging by confocal laser scanning microscopy,” Proc. Natl. Acad. Sci. U.S.A. 105(47), 18176–18181 (2008).
[CrossRef] [PubMed]

2007 (2)

D. L. Kolin and P. W. Wiseman, “Advances in image correlation spectroscopy: measuring number densities, aggregation states, and dynamics of fluorescently labeled macromolecules in cells,” Cell Biochem. Biophys. 49(3), 141–164 (2007).
[CrossRef] [PubMed]

C. M. Roth, P. I. Heinlein, M. Heilemann, and D.-P. Herten, “Imaging diffusion in living cells using time-correlated single-photon counting,” Anal. Chem. 79(19), 7340–7345 (2007).
[CrossRef] [PubMed]

2006 (4)

J. Beaudouin, F. Mora-Bermúdez, T. Klee, N. Daigle, and J. Ellenberg, “Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins,” Biophys. J. 90(6), 1878–1894 (2006).
[CrossRef] [PubMed]

C. Pack, K. Saito, M. Tamura, and M. Kinjo, “Microenvironment and effect of energy depletion in the nucleus analyzed by mobility of multiple oligomeric EGFPs,” Biophys. J. 91(10), 3921–3936 (2006).
[CrossRef] [PubMed]

D. L. Kolin, S. Costantino, and P. W. Wiseman, “Sampling effects, noise, and photobleaching in temporal image correlation spectroscopy,” Biophys. J. 90(2), 628–639 (2006).
[CrossRef]

D. L. Kolin, D. Ronis, and P. W. Wiseman, “k-Space image correlation spectroscopy: a method for accurate transport measurements independent of fluorophore photophysics,” Biophys. J. 91(8), 3061–3075 (2006).
[CrossRef] [PubMed]

2005 (3)

D. Grünwald, M. C. Cardoso, H. Leonhardt, and V. Buschmann, “Diffusion and binding properties investigated by Fluorescence Correlation Spectroscopy (FCS),” Curr. Pharm. Biotechnol. 6(5), 381–386 (2005).
[CrossRef] [PubMed]

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89(2), 1317–1327 (2005).
[CrossRef] [PubMed]

I. Gregor, D. Patra, and J. Enderlein, “Optical saturation in fluorescence correlation spectroscopy under continuous-wave and pulsed excitation,” ChemPhysChem 6(1), 164–170 (2005).
[CrossRef] [PubMed]

2003 (1)

W. B. Amos and J. G. White, “How the confocal laser scanning microscope entered biological research,” Biol. Cell 95(6), 335–342 (2003).
[CrossRef] [PubMed]

2001 (2)

P. Dittrich, F. Malvezzi-Campeggi, M. Jahnz, and P. Schwille, “Accessing molecular dynamics in cells by fluorescence correlation spectroscopy,” Biol. Chem. 382(3), 491–494 (2001).
[CrossRef] [PubMed]

G. S. Harms, L. Cognet, P. H. Lommerse, G. A. Blab, and T. Schmidt, “Autofluorescent proteins in single-molecule research: applications to live cell imaging microscopy,” Biophys. J. 80, 2396–2408 (2001).
[CrossRef] [PubMed]

2000 (1)

M. Wachsmuth, W. Waldeck, and J. Langowski, “Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy,” J. Mol. Biol. 298(4), 677–689 (2000).
[CrossRef] [PubMed]

1999 (1)

P. Schwille, U. Haupts, S. Maiti, and W. W. Webb, “Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation,” Biophys. J. 77(4), 2251–2265 (1999).
[CrossRef] [PubMed]

1996 (1)

K. M. Berland, P. T. C. So, Y. Chen, W. W. Mantulin, and E. Gratton, “Scanning two-photon fluctuation correlation spectroscopy: particle counting measurements for detection of molecular aggregation,” Biophys. J. 71(1), 410–420 (1996).
[CrossRef] [PubMed]

1995 (1)

J. Widengren, Ü. Mets, and R. Rigler, “Fluorescence Correlation Spectroscopy of Triplet States in Solution: A Theoretical and Experimental Study,” J. Phys. Chem. 99(36), 13368–13379 (1995).
[CrossRef]

1994 (1)

D. E. Koppel, F. Morgan, A. E. Cowan, and J. H. Carson, “Scanning concentration correlation spectroscopy using the confocal laser microscope,” Biophys. J. 66(2), 502–507 (1994).
[CrossRef] [PubMed]

1993 (1)

N. O. Petersen, P. L. Höddelius, P. W. Wiseman, O. Seger, and K. E. Magnusson, “Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application,” Biophys. J. 65(3), 1135–1146 (1993).
[CrossRef] [PubMed]

1978 (1)

C. Cremer and T. Cremer, “Considerations on a laser-scanning-microscope with high resolution and depth of field,” Microsc. Acta 81(1), 31–44 (1978).
[PubMed]

1974 (2)

E. L. Elson and D. Magde, “Fluorescence correlation spectroscopy. I. Conceptual basis and theory,” Biopolymers 13(1), 1–27 (1974).
[CrossRef]

D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974).
[CrossRef] [PubMed]

Amos, W. B.

W. B. Amos and J. G. White, “How the confocal laser scanning microscope entered biological research,” Biol. Cell 95(6), 335–342 (2003).
[CrossRef] [PubMed]

Bancaud, A.

A. Bancaud, S. Huet, N. Daigle, J. Mozziconacci, J. Beaudouin, and J. Ellenberg, “Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin,” EMBO J. 28(24), 3785–3798 (2009).
[CrossRef] [PubMed]

Beaudouin, J.

A. Bancaud, S. Huet, N. Daigle, J. Mozziconacci, J. Beaudouin, and J. Ellenberg, “Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin,” EMBO J. 28(24), 3785–3798 (2009).
[CrossRef] [PubMed]

K. P. Müller, F. Erdel, M. Caudron-Herger, C. Marth, B. D. Fodor, M. Richter, M. Scaranaro, J. Beaudouin, M. Wachsmuth, and K. Rippe, “Multiscale analysis of dynamics and interactions of heterochromatin protein 1 by fluorescence fluctuation microscopy,” Biophys. J. 97(11), 2876–2885 (2009).
[CrossRef] [PubMed]

J. Beaudouin, F. Mora-Bermúdez, T. Klee, N. Daigle, and J. Ellenberg, “Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins,” Biophys. J. 90(6), 1878–1894 (2006).
[CrossRef] [PubMed]

Berland, K. M.

K. M. Berland, P. T. C. So, Y. Chen, W. W. Mantulin, and E. Gratton, “Scanning two-photon fluctuation correlation spectroscopy: particle counting measurements for detection of molecular aggregation,” Biophys. J. 71(1), 410–420 (1996).
[CrossRef] [PubMed]

Blab, G. A.

G. S. Harms, L. Cognet, P. H. Lommerse, G. A. Blab, and T. Schmidt, “Autofluorescent proteins in single-molecule research: applications to live cell imaging microscopy,” Biophys. J. 80, 2396–2408 (2001).
[CrossRef] [PubMed]

Brown, C. M.

C. M. Brown, R. B. Dalal, B. Hebert, M. A. Digman, A. R. Horwitz, and E. Gratton, “Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope,” J. Microsc. 229(1), 78–91 (2008).
[CrossRef] [PubMed]

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89(2), 1317–1327 (2005).
[CrossRef] [PubMed]

Buschmann, V.

D. Grünwald, M. C. Cardoso, H. Leonhardt, and V. Buschmann, “Diffusion and binding properties investigated by Fluorescence Correlation Spectroscopy (FCS),” Curr. Pharm. Biotechnol. 6(5), 381–386 (2005).
[CrossRef] [PubMed]

Cardoso, M. C.

D. Grünwald, M. C. Cardoso, H. Leonhardt, and V. Buschmann, “Diffusion and binding properties investigated by Fluorescence Correlation Spectroscopy (FCS),” Curr. Pharm. Biotechnol. 6(5), 381–386 (2005).
[CrossRef] [PubMed]

Carson, J. H.

D. E. Koppel, F. Morgan, A. E. Cowan, and J. H. Carson, “Scanning concentration correlation spectroscopy using the confocal laser microscope,” Biophys. J. 66(2), 502–507 (1994).
[CrossRef] [PubMed]

Caudron-Herger, M.

K. P. Müller, F. Erdel, M. Caudron-Herger, C. Marth, B. D. Fodor, M. Richter, M. Scaranaro, J. Beaudouin, M. Wachsmuth, and K. Rippe, “Multiscale analysis of dynamics and interactions of heterochromatin protein 1 by fluorescence fluctuation microscopy,” Biophys. J. 97(11), 2876–2885 (2009).
[CrossRef] [PubMed]

M. Wachsmuth, M. Caudron-Herger, and K. Rippe, “Genome organization: balancing stability and plasticity,” Biochim. Biophys. Acta 1783(11), 2061–2079 (2008).
[CrossRef] [PubMed]

Chen, Y.

K. M. Berland, P. T. C. So, Y. Chen, W. W. Mantulin, and E. Gratton, “Scanning two-photon fluctuation correlation spectroscopy: particle counting measurements for detection of molecular aggregation,” Biophys. J. 71(1), 410–420 (1996).
[CrossRef] [PubMed]

Chiantia, S.

J. Ries, S. Chiantia, and P. Schwille, “Accurate determination of membrane dynamics with line-scan FCS,” Biophys. J. 96(5), 1999–2008 (2009).
[CrossRef] [PubMed]

Choi, C.

M. A. Digman, P. W. Wiseman, C. Choi, A. R. Horwitz, and E. Gratton, “Stoichiometry of molecular complexes at adhesions in living cells,” Proc. Natl. Acad. Sci. U.S.A. 106(7), 2170–2175 (2009).
[CrossRef] [PubMed]

Cognet, L.

G. S. Harms, L. Cognet, P. H. Lommerse, G. A. Blab, and T. Schmidt, “Autofluorescent proteins in single-molecule research: applications to live cell imaging microscopy,” Biophys. J. 80, 2396–2408 (2001).
[CrossRef] [PubMed]

Costantino, S.

D. L. Kolin, S. Costantino, and P. W. Wiseman, “Sampling effects, noise, and photobleaching in temporal image correlation spectroscopy,” Biophys. J. 90(2), 628–639 (2006).
[CrossRef]

Cowan, A. E.

D. E. Koppel, F. Morgan, A. E. Cowan, and J. H. Carson, “Scanning concentration correlation spectroscopy using the confocal laser microscope,” Biophys. J. 66(2), 502–507 (1994).
[CrossRef] [PubMed]

Cremer, C.

C. Cremer and T. Cremer, “Considerations on a laser-scanning-microscope with high resolution and depth of field,” Microsc. Acta 81(1), 31–44 (1978).
[PubMed]

Cremer, T.

C. Cremer and T. Cremer, “Considerations on a laser-scanning-microscope with high resolution and depth of field,” Microsc. Acta 81(1), 31–44 (1978).
[PubMed]

Daigle, N.

A. Bancaud, S. Huet, N. Daigle, J. Mozziconacci, J. Beaudouin, and J. Ellenberg, “Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin,” EMBO J. 28(24), 3785–3798 (2009).
[CrossRef] [PubMed]

J. Beaudouin, F. Mora-Bermúdez, T. Klee, N. Daigle, and J. Ellenberg, “Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins,” Biophys. J. 90(6), 1878–1894 (2006).
[CrossRef] [PubMed]

Dalal, R.

M. A. Digman, R. Dalal, A. F. Horwitz, and E. Gratton, “Mapping the number of molecules and brightness in the laser scanning microscope,” Biophys. J. 94(6), 2320–2332 (2008).
[CrossRef]

Dalal, R. B.

C. M. Brown, R. B. Dalal, B. Hebert, M. A. Digman, A. R. Horwitz, and E. Gratton, “Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope,” J. Microsc. 229(1), 78–91 (2008).
[CrossRef] [PubMed]

Digman, M. A.

M. A. Digman, P. W. Wiseman, C. Choi, A. R. Horwitz, and E. Gratton, “Stoichiometry of molecular complexes at adhesions in living cells,” Proc. Natl. Acad. Sci. U.S.A. 106(7), 2170–2175 (2009).
[CrossRef] [PubMed]

M. A. Digman and E. Gratton, “Analysis of diffusion and binding in cells using the RICS approach,” Microsc. Res. Tech. 72(4), 323–332 (2009).
[CrossRef]

M. A. Digman, R. Dalal, A. F. Horwitz, and E. Gratton, “Mapping the number of molecules and brightness in the laser scanning microscope,” Biophys. J. 94(6), 2320–2332 (2008).
[CrossRef]

C. M. Brown, R. B. Dalal, B. Hebert, M. A. Digman, A. R. Horwitz, and E. Gratton, “Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope,” J. Microsc. 229(1), 78–91 (2008).
[CrossRef] [PubMed]

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89(2), 1317–1327 (2005).
[CrossRef] [PubMed]

Dittrich, P.

P. Dittrich, F. Malvezzi-Campeggi, M. Jahnz, and P. Schwille, “Accessing molecular dynamics in cells by fluorescence correlation spectroscopy,” Biol. Chem. 382(3), 491–494 (2001).
[CrossRef] [PubMed]

Dross, N.

N. Dross, C. Spriet, M. Zwerger, G. Müller, W. Waldeck, J. Langowski, and J. Z. Rappoport, “Mapping eGFP oligomer mobility in living cell nuclei,” PLoS ONE 4(4), e5041–e5041 (2009).
[CrossRef] [PubMed]

Ellenberg, J.

A. Bancaud, S. Huet, N. Daigle, J. Mozziconacci, J. Beaudouin, and J. Ellenberg, “Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin,” EMBO J. 28(24), 3785–3798 (2009).
[CrossRef] [PubMed]

J. Beaudouin, F. Mora-Bermúdez, T. Klee, N. Daigle, and J. Ellenberg, “Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins,” Biophys. J. 90(6), 1878–1894 (2006).
[CrossRef] [PubMed]

Elson, E. L.

E. L. Elson and D. Magde, “Fluorescence correlation spectroscopy. I. Conceptual basis and theory,” Biopolymers 13(1), 1–27 (1974).
[CrossRef]

D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974).
[CrossRef] [PubMed]

Enderlein, J.

I. Gregor, D. Patra, and J. Enderlein, “Optical saturation in fluorescence correlation spectroscopy under continuous-wave and pulsed excitation,” ChemPhysChem 6(1), 164–170 (2005).
[CrossRef] [PubMed]

Erdel, F.

K. P. Müller, F. Erdel, M. Caudron-Herger, C. Marth, B. D. Fodor, M. Richter, M. Scaranaro, J. Beaudouin, M. Wachsmuth, and K. Rippe, “Multiscale analysis of dynamics and interactions of heterochromatin protein 1 by fluorescence fluctuation microscopy,” Biophys. J. 97(11), 2876–2885 (2009).
[CrossRef] [PubMed]

G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
[CrossRef] [PubMed]

Farla, P.

M. E. van Royen, P. Farla, K. A. Mattern, B. Geverts, J. Trapman, and A. B. Houtsmuller, “Fluorescence recovery after photobleaching (FRAP) to study nuclear protein dynamics in living cells,” Methods Mol. Biol. 464, 363–385 (2009).
[CrossRef]

Fodor, B. D.

K. P. Müller, F. Erdel, M. Caudron-Herger, C. Marth, B. D. Fodor, M. Richter, M. Scaranaro, J. Beaudouin, M. Wachsmuth, and K. Rippe, “Multiscale analysis of dynamics and interactions of heterochromatin protein 1 by fluorescence fluctuation microscopy,” Biophys. J. 97(11), 2876–2885 (2009).
[CrossRef] [PubMed]

Geverts, B.

M. E. van Royen, P. Farla, K. A. Mattern, B. Geverts, J. Trapman, and A. B. Houtsmuller, “Fluorescence recovery after photobleaching (FRAP) to study nuclear protein dynamics in living cells,” Methods Mol. Biol. 464, 363–385 (2009).
[CrossRef]

Gratton, E.

M. A. Digman and E. Gratton, “Analysis of diffusion and binding in cells using the RICS approach,” Microsc. Res. Tech. 72(4), 323–332 (2009).
[CrossRef]

M. A. Digman, P. W. Wiseman, C. Choi, A. R. Horwitz, and E. Gratton, “Stoichiometry of molecular complexes at adhesions in living cells,” Proc. Natl. Acad. Sci. U.S.A. 106(7), 2170–2175 (2009).
[CrossRef] [PubMed]

C. M. Brown, R. B. Dalal, B. Hebert, M. A. Digman, A. R. Horwitz, and E. Gratton, “Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope,” J. Microsc. 229(1), 78–91 (2008).
[CrossRef] [PubMed]

M. A. Digman, R. Dalal, A. F. Horwitz, and E. Gratton, “Mapping the number of molecules and brightness in the laser scanning microscope,” Biophys. J. 94(6), 2320–2332 (2008).
[CrossRef]

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89(2), 1317–1327 (2005).
[CrossRef] [PubMed]

K. M. Berland, P. T. C. So, Y. Chen, W. W. Mantulin, and E. Gratton, “Scanning two-photon fluctuation correlation spectroscopy: particle counting measurements for detection of molecular aggregation,” Biophys. J. 71(1), 410–420 (1996).
[CrossRef] [PubMed]

Gregor, I.

I. Gregor, D. Patra, and J. Enderlein, “Optical saturation in fluorescence correlation spectroscopy under continuous-wave and pulsed excitation,” ChemPhysChem 6(1), 164–170 (2005).
[CrossRef] [PubMed]

Grünwald, D.

D. Grünwald, M. C. Cardoso, H. Leonhardt, and V. Buschmann, “Diffusion and binding properties investigated by Fluorescence Correlation Spectroscopy (FCS),” Curr. Pharm. Biotechnol. 6(5), 381–386 (2005).
[CrossRef] [PubMed]

Harms, G. S.

G. S. Harms, L. Cognet, P. H. Lommerse, G. A. Blab, and T. Schmidt, “Autofluorescent proteins in single-molecule research: applications to live cell imaging microscopy,” Biophys. J. 80, 2396–2408 (2001).
[CrossRef] [PubMed]

Haupts, U.

P. Schwille, U. Haupts, S. Maiti, and W. W. Webb, “Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation,” Biophys. J. 77(4), 2251–2265 (1999).
[CrossRef] [PubMed]

Hebert, B.

C. M. Brown, R. B. Dalal, B. Hebert, M. A. Digman, A. R. Horwitz, and E. Gratton, “Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope,” J. Microsc. 229(1), 78–91 (2008).
[CrossRef] [PubMed]

Heidkamp, M.

V. Vukojević, M. Heidkamp, Y. Ming, B. Johansson, L. Terenius, and R. Rigler, “Quantitative single-molecule imaging by confocal laser scanning microscopy,” Proc. Natl. Acad. Sci. U.S.A. 105(47), 18176–18181 (2008).
[CrossRef] [PubMed]

Heilemann, M.

C. M. Roth, P. I. Heinlein, M. Heilemann, and D.-P. Herten, “Imaging diffusion in living cells using time-correlated single-photon counting,” Anal. Chem. 79(19), 7340–7345 (2007).
[CrossRef] [PubMed]

Heinlein, P. I.

C. M. Roth, P. I. Heinlein, M. Heilemann, and D.-P. Herten, “Imaging diffusion in living cells using time-correlated single-photon counting,” Anal. Chem. 79(19), 7340–7345 (2007).
[CrossRef] [PubMed]

Herten, D.-P.

C. M. Roth, P. I. Heinlein, M. Heilemann, and D.-P. Herten, “Imaging diffusion in living cells using time-correlated single-photon counting,” Anal. Chem. 79(19), 7340–7345 (2007).
[CrossRef] [PubMed]

Heuvelman, G.

G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
[CrossRef] [PubMed]

Höddelius, P. L.

N. O. Petersen, P. L. Höddelius, P. W. Wiseman, O. Seger, and K. E. Magnusson, “Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application,” Biophys. J. 65(3), 1135–1146 (1993).
[CrossRef] [PubMed]

Horwitz, A. F.

M. A. Digman, R. Dalal, A. F. Horwitz, and E. Gratton, “Mapping the number of molecules and brightness in the laser scanning microscope,” Biophys. J. 94(6), 2320–2332 (2008).
[CrossRef]

Horwitz, A. R.

M. A. Digman, P. W. Wiseman, C. Choi, A. R. Horwitz, and E. Gratton, “Stoichiometry of molecular complexes at adhesions in living cells,” Proc. Natl. Acad. Sci. U.S.A. 106(7), 2170–2175 (2009).
[CrossRef] [PubMed]

C. M. Brown, R. B. Dalal, B. Hebert, M. A. Digman, A. R. Horwitz, and E. Gratton, “Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope,” J. Microsc. 229(1), 78–91 (2008).
[CrossRef] [PubMed]

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89(2), 1317–1327 (2005).
[CrossRef] [PubMed]

Houtsmuller, A. B.

M. E. van Royen, P. Farla, K. A. Mattern, B. Geverts, J. Trapman, and A. B. Houtsmuller, “Fluorescence recovery after photobleaching (FRAP) to study nuclear protein dynamics in living cells,” Methods Mol. Biol. 464, 363–385 (2009).
[CrossRef]

Huet, S.

A. Bancaud, S. Huet, N. Daigle, J. Mozziconacci, J. Beaudouin, and J. Ellenberg, “Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin,” EMBO J. 28(24), 3785–3798 (2009).
[CrossRef] [PubMed]

Jahnz, M.

P. Dittrich, F. Malvezzi-Campeggi, M. Jahnz, and P. Schwille, “Accessing molecular dynamics in cells by fluorescence correlation spectroscopy,” Biol. Chem. 382(3), 491–494 (2001).
[CrossRef] [PubMed]

Johansson, B.

V. Vukojević, M. Heidkamp, Y. Ming, B. Johansson, L. Terenius, and R. Rigler, “Quantitative single-molecule imaging by confocal laser scanning microscopy,” Proc. Natl. Acad. Sci. U.S.A. 105(47), 18176–18181 (2008).
[CrossRef] [PubMed]

Kinjo, M.

C. Pack, K. Saito, M. Tamura, and M. Kinjo, “Microenvironment and effect of energy depletion in the nucleus analyzed by mobility of multiple oligomeric EGFPs,” Biophys. J. 91(10), 3921–3936 (2006).
[CrossRef] [PubMed]

Klee, T.

J. Beaudouin, F. Mora-Bermúdez, T. Klee, N. Daigle, and J. Ellenberg, “Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins,” Biophys. J. 90(6), 1878–1894 (2006).
[CrossRef] [PubMed]

Kolin, D. L.

D. L. Kolin and P. W. Wiseman, “Advances in image correlation spectroscopy: measuring number densities, aggregation states, and dynamics of fluorescently labeled macromolecules in cells,” Cell Biochem. Biophys. 49(3), 141–164 (2007).
[CrossRef] [PubMed]

D. L. Kolin, D. Ronis, and P. W. Wiseman, “k-Space image correlation spectroscopy: a method for accurate transport measurements independent of fluorophore photophysics,” Biophys. J. 91(8), 3061–3075 (2006).
[CrossRef] [PubMed]

D. L. Kolin, S. Costantino, and P. W. Wiseman, “Sampling effects, noise, and photobleaching in temporal image correlation spectroscopy,” Biophys. J. 90(2), 628–639 (2006).
[CrossRef]

Koppel, D. E.

D. E. Koppel, F. Morgan, A. E. Cowan, and J. H. Carson, “Scanning concentration correlation spectroscopy using the confocal laser microscope,” Biophys. J. 66(2), 502–507 (1994).
[CrossRef] [PubMed]

Langowski, J.

N. Dross, C. Spriet, M. Zwerger, G. Müller, W. Waldeck, J. Langowski, and J. Z. Rappoport, “Mapping eGFP oligomer mobility in living cell nuclei,” PLoS ONE 4(4), e5041–e5041 (2009).
[CrossRef] [PubMed]

M. Wachsmuth, W. Waldeck, and J. Langowski, “Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy,” J. Mol. Biol. 298(4), 677–689 (2000).
[CrossRef] [PubMed]

Leonhardt, H.

D. Grünwald, M. C. Cardoso, H. Leonhardt, and V. Buschmann, “Diffusion and binding properties investigated by Fluorescence Correlation Spectroscopy (FCS),” Curr. Pharm. Biotechnol. 6(5), 381–386 (2005).
[CrossRef] [PubMed]

Lommerse, P. H.

G. S. Harms, L. Cognet, P. H. Lommerse, G. A. Blab, and T. Schmidt, “Autofluorescent proteins in single-molecule research: applications to live cell imaging microscopy,” Biophys. J. 80, 2396–2408 (2001).
[CrossRef] [PubMed]

Magde, D.

E. L. Elson and D. Magde, “Fluorescence correlation spectroscopy. I. Conceptual basis and theory,” Biopolymers 13(1), 1–27 (1974).
[CrossRef]

D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974).
[CrossRef] [PubMed]

Magnusson, K. E.

N. O. Petersen, P. L. Höddelius, P. W. Wiseman, O. Seger, and K. E. Magnusson, “Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application,” Biophys. J. 65(3), 1135–1146 (1993).
[CrossRef] [PubMed]

Maiti, S.

P. Schwille, U. Haupts, S. Maiti, and W. W. Webb, “Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation,” Biophys. J. 77(4), 2251–2265 (1999).
[CrossRef] [PubMed]

Malvezzi-Campeggi, F.

P. Dittrich, F. Malvezzi-Campeggi, M. Jahnz, and P. Schwille, “Accessing molecular dynamics in cells by fluorescence correlation spectroscopy,” Biol. Chem. 382(3), 491–494 (2001).
[CrossRef] [PubMed]

Mantulin, W. W.

K. M. Berland, P. T. C. So, Y. Chen, W. W. Mantulin, and E. Gratton, “Scanning two-photon fluctuation correlation spectroscopy: particle counting measurements for detection of molecular aggregation,” Biophys. J. 71(1), 410–420 (1996).
[CrossRef] [PubMed]

Marth, C.

K. P. Müller, F. Erdel, M. Caudron-Herger, C. Marth, B. D. Fodor, M. Richter, M. Scaranaro, J. Beaudouin, M. Wachsmuth, and K. Rippe, “Multiscale analysis of dynamics and interactions of heterochromatin protein 1 by fluorescence fluctuation microscopy,” Biophys. J. 97(11), 2876–2885 (2009).
[CrossRef] [PubMed]

Mattern, K. A.

M. E. van Royen, P. Farla, K. A. Mattern, B. Geverts, J. Trapman, and A. B. Houtsmuller, “Fluorescence recovery after photobleaching (FRAP) to study nuclear protein dynamics in living cells,” Methods Mol. Biol. 464, 363–385 (2009).
[CrossRef]

Mets, Ü.

J. Widengren, Ü. Mets, and R. Rigler, “Fluorescence Correlation Spectroscopy of Triplet States in Solution: A Theoretical and Experimental Study,” J. Phys. Chem. 99(36), 13368–13379 (1995).
[CrossRef]

Ming, Y.

V. Vukojević, M. Heidkamp, Y. Ming, B. Johansson, L. Terenius, and R. Rigler, “Quantitative single-molecule imaging by confocal laser scanning microscopy,” Proc. Natl. Acad. Sci. U.S.A. 105(47), 18176–18181 (2008).
[CrossRef] [PubMed]

Mitchison, T. J.

D. J. Needleman, Y. Xu, and T. J. Mitchison, “Pin-hole array correlation imaging: highly parallel fluorescence correlation spectroscopy,” Biophys. J. 96(12), 5050–5059 (2009).
[CrossRef] [PubMed]

Mora-Bermúdez, F.

J. Beaudouin, F. Mora-Bermúdez, T. Klee, N. Daigle, and J. Ellenberg, “Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins,” Biophys. J. 90(6), 1878–1894 (2006).
[CrossRef] [PubMed]

Morgan, F.

D. E. Koppel, F. Morgan, A. E. Cowan, and J. H. Carson, “Scanning concentration correlation spectroscopy using the confocal laser microscope,” Biophys. J. 66(2), 502–507 (1994).
[CrossRef] [PubMed]

Mozziconacci, J.

A. Bancaud, S. Huet, N. Daigle, J. Mozziconacci, J. Beaudouin, and J. Ellenberg, “Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin,” EMBO J. 28(24), 3785–3798 (2009).
[CrossRef] [PubMed]

Müller, G.

N. Dross, C. Spriet, M. Zwerger, G. Müller, W. Waldeck, J. Langowski, and J. Z. Rappoport, “Mapping eGFP oligomer mobility in living cell nuclei,” PLoS ONE 4(4), e5041–e5041 (2009).
[CrossRef] [PubMed]

Müller, K. P.

K. P. Müller, F. Erdel, M. Caudron-Herger, C. Marth, B. D. Fodor, M. Richter, M. Scaranaro, J. Beaudouin, M. Wachsmuth, and K. Rippe, “Multiscale analysis of dynamics and interactions of heterochromatin protein 1 by fluorescence fluctuation microscopy,” Biophys. J. 97(11), 2876–2885 (2009).
[CrossRef] [PubMed]

Needleman, D. J.

D. J. Needleman, Y. Xu, and T. J. Mitchison, “Pin-hole array correlation imaging: highly parallel fluorescence correlation spectroscopy,” Biophys. J. 96(12), 5050–5059 (2009).
[CrossRef] [PubMed]

Pack, C.

C. Pack, K. Saito, M. Tamura, and M. Kinjo, “Microenvironment and effect of energy depletion in the nucleus analyzed by mobility of multiple oligomeric EGFPs,” Biophys. J. 91(10), 3921–3936 (2006).
[CrossRef] [PubMed]

Patra, D.

I. Gregor, D. Patra, and J. Enderlein, “Optical saturation in fluorescence correlation spectroscopy under continuous-wave and pulsed excitation,” ChemPhysChem 6(1), 164–170 (2005).
[CrossRef] [PubMed]

Petersen, N. O.

N. O. Petersen, P. L. Höddelius, P. W. Wiseman, O. Seger, and K. E. Magnusson, “Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application,” Biophys. J. 65(3), 1135–1146 (1993).
[CrossRef] [PubMed]

Rappoport, J. Z.

N. Dross, C. Spriet, M. Zwerger, G. Müller, W. Waldeck, J. Langowski, and J. Z. Rappoport, “Mapping eGFP oligomer mobility in living cell nuclei,” PLoS ONE 4(4), e5041–e5041 (2009).
[CrossRef] [PubMed]

Richter, M.

K. P. Müller, F. Erdel, M. Caudron-Herger, C. Marth, B. D. Fodor, M. Richter, M. Scaranaro, J. Beaudouin, M. Wachsmuth, and K. Rippe, “Multiscale analysis of dynamics and interactions of heterochromatin protein 1 by fluorescence fluctuation microscopy,” Biophys. J. 97(11), 2876–2885 (2009).
[CrossRef] [PubMed]

Ries, J.

J. Ries, S. Chiantia, and P. Schwille, “Accurate determination of membrane dynamics with line-scan FCS,” Biophys. J. 96(5), 1999–2008 (2009).
[CrossRef] [PubMed]

Rigler, R.

V. Vukojević, M. Heidkamp, Y. Ming, B. Johansson, L. Terenius, and R. Rigler, “Quantitative single-molecule imaging by confocal laser scanning microscopy,” Proc. Natl. Acad. Sci. U.S.A. 105(47), 18176–18181 (2008).
[CrossRef] [PubMed]

J. Widengren, Ü. Mets, and R. Rigler, “Fluorescence Correlation Spectroscopy of Triplet States in Solution: A Theoretical and Experimental Study,” J. Phys. Chem. 99(36), 13368–13379 (1995).
[CrossRef]

Rippe, K.

K. P. Müller, F. Erdel, M. Caudron-Herger, C. Marth, B. D. Fodor, M. Richter, M. Scaranaro, J. Beaudouin, M. Wachsmuth, and K. Rippe, “Multiscale analysis of dynamics and interactions of heterochromatin protein 1 by fluorescence fluctuation microscopy,” Biophys. J. 97(11), 2876–2885 (2009).
[CrossRef] [PubMed]

G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
[CrossRef] [PubMed]

M. Wachsmuth, M. Caudron-Herger, and K. Rippe, “Genome organization: balancing stability and plasticity,” Biochim. Biophys. Acta 1783(11), 2061–2079 (2008).
[CrossRef] [PubMed]

Ronis, D.

D. L. Kolin, D. Ronis, and P. W. Wiseman, “k-Space image correlation spectroscopy: a method for accurate transport measurements independent of fluorophore photophysics,” Biophys. J. 91(8), 3061–3075 (2006).
[CrossRef] [PubMed]

Roth, C. M.

C. M. Roth, P. I. Heinlein, M. Heilemann, and D.-P. Herten, “Imaging diffusion in living cells using time-correlated single-photon counting,” Anal. Chem. 79(19), 7340–7345 (2007).
[CrossRef] [PubMed]

Saito, K.

C. Pack, K. Saito, M. Tamura, and M. Kinjo, “Microenvironment and effect of energy depletion in the nucleus analyzed by mobility of multiple oligomeric EGFPs,” Biophys. J. 91(10), 3921–3936 (2006).
[CrossRef] [PubMed]

Scaranaro, M.

K. P. Müller, F. Erdel, M. Caudron-Herger, C. Marth, B. D. Fodor, M. Richter, M. Scaranaro, J. Beaudouin, M. Wachsmuth, and K. Rippe, “Multiscale analysis of dynamics and interactions of heterochromatin protein 1 by fluorescence fluctuation microscopy,” Biophys. J. 97(11), 2876–2885 (2009).
[CrossRef] [PubMed]

Schmidt, T.

G. S. Harms, L. Cognet, P. H. Lommerse, G. A. Blab, and T. Schmidt, “Autofluorescent proteins in single-molecule research: applications to live cell imaging microscopy,” Biophys. J. 80, 2396–2408 (2001).
[CrossRef] [PubMed]

Schwille, P.

J. Ries, S. Chiantia, and P. Schwille, “Accurate determination of membrane dynamics with line-scan FCS,” Biophys. J. 96(5), 1999–2008 (2009).
[CrossRef] [PubMed]

P. Dittrich, F. Malvezzi-Campeggi, M. Jahnz, and P. Schwille, “Accessing molecular dynamics in cells by fluorescence correlation spectroscopy,” Biol. Chem. 382(3), 491–494 (2001).
[CrossRef] [PubMed]

P. Schwille, U. Haupts, S. Maiti, and W. W. Webb, “Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation,” Biophys. J. 77(4), 2251–2265 (1999).
[CrossRef] [PubMed]

Seger, O.

N. O. Petersen, P. L. Höddelius, P. W. Wiseman, O. Seger, and K. E. Magnusson, “Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application,” Biophys. J. 65(3), 1135–1146 (1993).
[CrossRef] [PubMed]

Sengupta, P.

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89(2), 1317–1327 (2005).
[CrossRef] [PubMed]

So, P. T. C.

K. M. Berland, P. T. C. So, Y. Chen, W. W. Mantulin, and E. Gratton, “Scanning two-photon fluctuation correlation spectroscopy: particle counting measurements for detection of molecular aggregation,” Biophys. J. 71(1), 410–420 (1996).
[CrossRef] [PubMed]

Spriet, C.

N. Dross, C. Spriet, M. Zwerger, G. Müller, W. Waldeck, J. Langowski, and J. Z. Rappoport, “Mapping eGFP oligomer mobility in living cell nuclei,” PLoS ONE 4(4), e5041–e5041 (2009).
[CrossRef] [PubMed]

Tamura, M.

C. Pack, K. Saito, M. Tamura, and M. Kinjo, “Microenvironment and effect of energy depletion in the nucleus analyzed by mobility of multiple oligomeric EGFPs,” Biophys. J. 91(10), 3921–3936 (2006).
[CrossRef] [PubMed]

Terenius, L.

V. Vukojević, M. Heidkamp, Y. Ming, B. Johansson, L. Terenius, and R. Rigler, “Quantitative single-molecule imaging by confocal laser scanning microscopy,” Proc. Natl. Acad. Sci. U.S.A. 105(47), 18176–18181 (2008).
[CrossRef] [PubMed]

Trapman, J.

M. E. van Royen, P. Farla, K. A. Mattern, B. Geverts, J. Trapman, and A. B. Houtsmuller, “Fluorescence recovery after photobleaching (FRAP) to study nuclear protein dynamics in living cells,” Methods Mol. Biol. 464, 363–385 (2009).
[CrossRef]

van Royen, M. E.

M. E. van Royen, P. Farla, K. A. Mattern, B. Geverts, J. Trapman, and A. B. Houtsmuller, “Fluorescence recovery after photobleaching (FRAP) to study nuclear protein dynamics in living cells,” Methods Mol. Biol. 464, 363–385 (2009).
[CrossRef]

Vukojevic, V.

V. Vukojević, M. Heidkamp, Y. Ming, B. Johansson, L. Terenius, and R. Rigler, “Quantitative single-molecule imaging by confocal laser scanning microscopy,” Proc. Natl. Acad. Sci. U.S.A. 105(47), 18176–18181 (2008).
[CrossRef] [PubMed]

Wachsmuth, M.

G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
[CrossRef] [PubMed]

K. P. Müller, F. Erdel, M. Caudron-Herger, C. Marth, B. D. Fodor, M. Richter, M. Scaranaro, J. Beaudouin, M. Wachsmuth, and K. Rippe, “Multiscale analysis of dynamics and interactions of heterochromatin protein 1 by fluorescence fluctuation microscopy,” Biophys. J. 97(11), 2876–2885 (2009).
[CrossRef] [PubMed]

M. Wachsmuth, M. Caudron-Herger, and K. Rippe, “Genome organization: balancing stability and plasticity,” Biochim. Biophys. Acta 1783(11), 2061–2079 (2008).
[CrossRef] [PubMed]

M. Wachsmuth, W. Waldeck, and J. Langowski, “Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy,” J. Mol. Biol. 298(4), 677–689 (2000).
[CrossRef] [PubMed]

Waldeck, W.

N. Dross, C. Spriet, M. Zwerger, G. Müller, W. Waldeck, J. Langowski, and J. Z. Rappoport, “Mapping eGFP oligomer mobility in living cell nuclei,” PLoS ONE 4(4), e5041–e5041 (2009).
[CrossRef] [PubMed]

M. Wachsmuth, W. Waldeck, and J. Langowski, “Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy,” J. Mol. Biol. 298(4), 677–689 (2000).
[CrossRef] [PubMed]

Webb, W. W.

P. Schwille, U. Haupts, S. Maiti, and W. W. Webb, “Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation,” Biophys. J. 77(4), 2251–2265 (1999).
[CrossRef] [PubMed]

D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974).
[CrossRef] [PubMed]

White, J. G.

W. B. Amos and J. G. White, “How the confocal laser scanning microscope entered biological research,” Biol. Cell 95(6), 335–342 (2003).
[CrossRef] [PubMed]

Widengren, J.

J. Widengren, Ü. Mets, and R. Rigler, “Fluorescence Correlation Spectroscopy of Triplet States in Solution: A Theoretical and Experimental Study,” J. Phys. Chem. 99(36), 13368–13379 (1995).
[CrossRef]

Wiseman, P. W.

M. A. Digman, P. W. Wiseman, C. Choi, A. R. Horwitz, and E. Gratton, “Stoichiometry of molecular complexes at adhesions in living cells,” Proc. Natl. Acad. Sci. U.S.A. 106(7), 2170–2175 (2009).
[CrossRef] [PubMed]

D. L. Kolin and P. W. Wiseman, “Advances in image correlation spectroscopy: measuring number densities, aggregation states, and dynamics of fluorescently labeled macromolecules in cells,” Cell Biochem. Biophys. 49(3), 141–164 (2007).
[CrossRef] [PubMed]

D. L. Kolin, D. Ronis, and P. W. Wiseman, “k-Space image correlation spectroscopy: a method for accurate transport measurements independent of fluorophore photophysics,” Biophys. J. 91(8), 3061–3075 (2006).
[CrossRef] [PubMed]

D. L. Kolin, S. Costantino, and P. W. Wiseman, “Sampling effects, noise, and photobleaching in temporal image correlation spectroscopy,” Biophys. J. 90(2), 628–639 (2006).
[CrossRef]

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89(2), 1317–1327 (2005).
[CrossRef] [PubMed]

N. O. Petersen, P. L. Höddelius, P. W. Wiseman, O. Seger, and K. E. Magnusson, “Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application,” Biophys. J. 65(3), 1135–1146 (1993).
[CrossRef] [PubMed]

Xu, Y.

D. J. Needleman, Y. Xu, and T. J. Mitchison, “Pin-hole array correlation imaging: highly parallel fluorescence correlation spectroscopy,” Biophys. J. 96(12), 5050–5059 (2009).
[CrossRef] [PubMed]

Zwerger, M.

N. Dross, C. Spriet, M. Zwerger, G. Müller, W. Waldeck, J. Langowski, and J. Z. Rappoport, “Mapping eGFP oligomer mobility in living cell nuclei,” PLoS ONE 4(4), e5041–e5041 (2009).
[CrossRef] [PubMed]

Anal. Chem. (1)

C. M. Roth, P. I. Heinlein, M. Heilemann, and D.-P. Herten, “Imaging diffusion in living cells using time-correlated single-photon counting,” Anal. Chem. 79(19), 7340–7345 (2007).
[CrossRef] [PubMed]

Biochim. Biophys. Acta (1)

M. Wachsmuth, M. Caudron-Herger, and K. Rippe, “Genome organization: balancing stability and plasticity,” Biochim. Biophys. Acta 1783(11), 2061–2079 (2008).
[CrossRef] [PubMed]

Biol. Cell (1)

W. B. Amos and J. G. White, “How the confocal laser scanning microscope entered biological research,” Biol. Cell 95(6), 335–342 (2003).
[CrossRef] [PubMed]

Biol. Chem. (1)

P. Dittrich, F. Malvezzi-Campeggi, M. Jahnz, and P. Schwille, “Accessing molecular dynamics in cells by fluorescence correlation spectroscopy,” Biol. Chem. 382(3), 491–494 (2001).
[CrossRef] [PubMed]

Biophys. J. (14)

P. Schwille, U. Haupts, S. Maiti, and W. W. Webb, “Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation,” Biophys. J. 77(4), 2251–2265 (1999).
[CrossRef] [PubMed]

K. P. Müller, F. Erdel, M. Caudron-Herger, C. Marth, B. D. Fodor, M. Richter, M. Scaranaro, J. Beaudouin, M. Wachsmuth, and K. Rippe, “Multiscale analysis of dynamics and interactions of heterochromatin protein 1 by fluorescence fluctuation microscopy,” Biophys. J. 97(11), 2876–2885 (2009).
[CrossRef] [PubMed]

J. Beaudouin, F. Mora-Bermúdez, T. Klee, N. Daigle, and J. Ellenberg, “Dissecting the contribution of diffusion and interactions to the mobility of nuclear proteins,” Biophys. J. 90(6), 1878–1894 (2006).
[CrossRef] [PubMed]

C. Pack, K. Saito, M. Tamura, and M. Kinjo, “Microenvironment and effect of energy depletion in the nucleus analyzed by mobility of multiple oligomeric EGFPs,” Biophys. J. 91(10), 3921–3936 (2006).
[CrossRef] [PubMed]

G. S. Harms, L. Cognet, P. H. Lommerse, G. A. Blab, and T. Schmidt, “Autofluorescent proteins in single-molecule research: applications to live cell imaging microscopy,” Biophys. J. 80, 2396–2408 (2001).
[CrossRef] [PubMed]

N. O. Petersen, P. L. Höddelius, P. W. Wiseman, O. Seger, and K. E. Magnusson, “Quantitation of membrane receptor distributions by image correlation spectroscopy: concept and application,” Biophys. J. 65(3), 1135–1146 (1993).
[CrossRef] [PubMed]

M. A. Digman, R. Dalal, A. F. Horwitz, and E. Gratton, “Mapping the number of molecules and brightness in the laser scanning microscope,” Biophys. J. 94(6), 2320–2332 (2008).
[CrossRef]

K. M. Berland, P. T. C. So, Y. Chen, W. W. Mantulin, and E. Gratton, “Scanning two-photon fluctuation correlation spectroscopy: particle counting measurements for detection of molecular aggregation,” Biophys. J. 71(1), 410–420 (1996).
[CrossRef] [PubMed]

D. E. Koppel, F. Morgan, A. E. Cowan, and J. H. Carson, “Scanning concentration correlation spectroscopy using the confocal laser microscope,” Biophys. J. 66(2), 502–507 (1994).
[CrossRef] [PubMed]

J. Ries, S. Chiantia, and P. Schwille, “Accurate determination of membrane dynamics with line-scan FCS,” Biophys. J. 96(5), 1999–2008 (2009).
[CrossRef] [PubMed]

D. L. Kolin, S. Costantino, and P. W. Wiseman, “Sampling effects, noise, and photobleaching in temporal image correlation spectroscopy,” Biophys. J. 90(2), 628–639 (2006).
[CrossRef]

D. L. Kolin, D. Ronis, and P. W. Wiseman, “k-Space image correlation spectroscopy: a method for accurate transport measurements independent of fluorophore photophysics,” Biophys. J. 91(8), 3061–3075 (2006).
[CrossRef] [PubMed]

M. A. Digman, C. M. Brown, P. Sengupta, P. W. Wiseman, A. R. Horwitz, and E. Gratton, “Measuring fast dynamics in solutions and cells with a laser scanning microscope,” Biophys. J. 89(2), 1317–1327 (2005).
[CrossRef] [PubMed]

D. J. Needleman, Y. Xu, and T. J. Mitchison, “Pin-hole array correlation imaging: highly parallel fluorescence correlation spectroscopy,” Biophys. J. 96(12), 5050–5059 (2009).
[CrossRef] [PubMed]

Biopolymers (2)

E. L. Elson and D. Magde, “Fluorescence correlation spectroscopy. I. Conceptual basis and theory,” Biopolymers 13(1), 1–27 (1974).
[CrossRef]

D. Magde, E. L. Elson, and W. W. Webb, “Fluorescence correlation spectroscopy. II. An experimental realization,” Biopolymers 13(1), 29–61 (1974).
[CrossRef] [PubMed]

Cell Biochem. Biophys. (1)

D. L. Kolin and P. W. Wiseman, “Advances in image correlation spectroscopy: measuring number densities, aggregation states, and dynamics of fluorescently labeled macromolecules in cells,” Cell Biochem. Biophys. 49(3), 141–164 (2007).
[CrossRef] [PubMed]

ChemPhysChem (1)

I. Gregor, D. Patra, and J. Enderlein, “Optical saturation in fluorescence correlation spectroscopy under continuous-wave and pulsed excitation,” ChemPhysChem 6(1), 164–170 (2005).
[CrossRef] [PubMed]

Curr. Pharm. Biotechnol. (1)

D. Grünwald, M. C. Cardoso, H. Leonhardt, and V. Buschmann, “Diffusion and binding properties investigated by Fluorescence Correlation Spectroscopy (FCS),” Curr. Pharm. Biotechnol. 6(5), 381–386 (2005).
[CrossRef] [PubMed]

EMBO J. (1)

A. Bancaud, S. Huet, N. Daigle, J. Mozziconacci, J. Beaudouin, and J. Ellenberg, “Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin,” EMBO J. 28(24), 3785–3798 (2009).
[CrossRef] [PubMed]

Eur. Biophys. J. (1)

G. Heuvelman, F. Erdel, M. Wachsmuth, and K. Rippe, “Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy,” Eur. Biophys. J. 38(6), 813–828 (2009).
[CrossRef] [PubMed]

J. Microsc. (1)

C. M. Brown, R. B. Dalal, B. Hebert, M. A. Digman, A. R. Horwitz, and E. Gratton, “Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope,” J. Microsc. 229(1), 78–91 (2008).
[CrossRef] [PubMed]

J. Mol. Biol. (1)

M. Wachsmuth, W. Waldeck, and J. Langowski, “Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy,” J. Mol. Biol. 298(4), 677–689 (2000).
[CrossRef] [PubMed]

J. Phys. Chem. (1)

J. Widengren, Ü. Mets, and R. Rigler, “Fluorescence Correlation Spectroscopy of Triplet States in Solution: A Theoretical and Experimental Study,” J. Phys. Chem. 99(36), 13368–13379 (1995).
[CrossRef]

Methods Mol. Biol. (1)

M. E. van Royen, P. Farla, K. A. Mattern, B. Geverts, J. Trapman, and A. B. Houtsmuller, “Fluorescence recovery after photobleaching (FRAP) to study nuclear protein dynamics in living cells,” Methods Mol. Biol. 464, 363–385 (2009).
[CrossRef]

Microsc. Acta (1)

C. Cremer and T. Cremer, “Considerations on a laser-scanning-microscope with high resolution and depth of field,” Microsc. Acta 81(1), 31–44 (1978).
[PubMed]

Microsc. Res. Tech. (1)

M. A. Digman and E. Gratton, “Analysis of diffusion and binding in cells using the RICS approach,” Microsc. Res. Tech. 72(4), 323–332 (2009).
[CrossRef]

PLoS ONE (1)

N. Dross, C. Spriet, M. Zwerger, G. Müller, W. Waldeck, J. Langowski, and J. Z. Rappoport, “Mapping eGFP oligomer mobility in living cell nuclei,” PLoS ONE 4(4), e5041–e5041 (2009).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. U.S.A. (2)

M. A. Digman, P. W. Wiseman, C. Choi, A. R. Horwitz, and E. Gratton, “Stoichiometry of molecular complexes at adhesions in living cells,” Proc. Natl. Acad. Sci. U.S.A. 106(7), 2170–2175 (2009).
[CrossRef] [PubMed]

V. Vukojević, M. Heidkamp, Y. Ming, B. Johansson, L. Terenius, and R. Rigler, “Quantitative single-molecule imaging by confocal laser scanning microscopy,” Proc. Natl. Acad. Sci. U.S.A. 105(47), 18176–18181 (2008).
[CrossRef] [PubMed]

Other (10)

N. O. Petersen, R. Rigler, and E. S. Elson, “FCS and spatial correlations on biological surfaces,” in Fluorescence Correlation Spectroscopy - Theory and Applications (Springer, Heidelberg, 2001), pp. 162–184.

“LFD Workshop 2006 - Laboratory for Fluorescence Dynamics” (2006), http://www.lfd.uci.edu/workshop/2006/ .

M. Wachsmuth, “Method for measuring fluorescence fluctuations in the presence of slow signal fluctuations,” US Patent No. 7,154,602 (2006).

J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Kluwer Academic/Plenum Publishers, New York, 1999).

E. Gielen, N. Smisdom, M. Vandeven, B. De Clercq, E. Gratton, M. Digman, J.-M. Rigo, J. Hofkens, Y. Engelborghs, and M. Ameloot, “Measuring Diffusion of Lipid-like Probes in Artificial and Natural Membranes by Raster Image Correlation Spectroscopy (RICS): Use of a Commercial Laser-Scanning Microscope with Analog Detection,” Langmuir (2009).

J. Pawley, Handbook of Biological Confocal Microscopy (Springer, Berlin, 2006).

G. Rabut, J. Ellenberg, D. Spector, and D. Goldman, “Photobleaching Techniques to Study Mobility and Molecular Dynamics of Proteins in Live Cells: FRAP, iFRAP, and FLIP,” in Live Cell Imaging - A Laboratory Manual (CSHL Press, Cold Spring Harbor, 2005), pp. 101–126.

M. Wachsmuth, and K. Weisshart, “Fluorescence photobleaching and fluorescence correlation spectroscopy: two complementary technologies to study molecular dynamics in living cells,” in Imaging Cellular and Molecular Biological Functions (Springer Verlag, Heidelberg, 2007).

D. A. Bulseco, D. E. Wolf, S. Greenfield, and E. W. David, “Fluorescence Correlation Spectroscopy: Molecular Complexing in Solution and in Living Cells,” in Digital Microscopy, 3rd Edition (Academic Press, 2007), pp. 525–559.

B. Kannan, L. Guo, T. Sudhaharan, S. Ahmed, I. Maruyama, and T. Wohland, “Spatially Resolved Total Internal Reflection Fluorescence Correlation Microscopy Using an Electron Multiplying Charge-Coupled Device Camera,” Analytical Chemistry (2007).

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (6)

Fig. 1
Fig. 1

Principle of RICS and msICS. (a) Confocal images scanned unidirectionally with well defined scanning velocity, pixel size, pixel dwell time and line scan time are subject to a two-dimensional spatial autocorrelation analysis. (b) For RICS, a two-dimensional autocorrelation function for a given scanning velocity is generated and fitted with a model function describing transport/diffusion, photophysical and interaction process to extract the spatio-temporal information inscribed into the image by the scanning process. (c) For msICS, a one-dimensional representation of the resulting autocorrelation function for a given pixel shift is generated as a function of the lag time given by the pixel dwell time for smaller and by the line time for larger times resulting from different scanning velocities and is fitted accordingly.

Fig. 2
Fig. 2

Characterization of the PSF. (a) Mean fluorescence intensity of Alexa488 taken from confocal images (circles, left axis) and the fit of Eq. (7) (red line) as well as the focal radius normalized to the value determined at 30 μW (squares, right axis) as a function of incident laser intensity. The vertical line marks the saturation power. (b) Map of the focal radius w 0 and (c) of the focal volume V 0 as determined with FCS of Alexa488 in solution on a 9 × 9 array of points covering an area of 243 × 243 μm2. The variations are very small within the central 135 × 135 μm2 area where the experiments were carried out.

Fig. 3
Fig. 3

RICS in a living cell. (a) Detail of a confocal image of a HeLa cell expressing EGFP with a ROI in the nucleolus (white square; 50×50 pixels, 1.5×1.5 μm2) used for RICS analysis (scale bar 5 μm): (b) autocorrelation function averaged over 7 images acquired at 35 Hz scanning frequency (circles) and fit (line) of Eq. (2) yielding a diffusion coefficent of 23 μm2 s–1.

Fig. 4
Fig. 4

msICS in solution. (a) msICS, ξ = 2 , of Alexa488 (20 nM) from a ROI of 200×200 pixels and 6×6 μm2 (circles) and fit (line) with Eq. (2). (b) msICS, ξ = 1 10 (circles, decreasing amplitude with increasing ξ), of the same sample and ROI with global fit (lines). (c) Point FCS of the same sample (circles) and fit (line) with Eq. (7). For fit results see Table 2.

Fig. 5
Fig. 5

Two-dimensional graph of the autocorrelation functions of data acquired in the nucleolus of a HeLa cell expressing EGFP (ROI: 100×100 pixels, 3×3 μm2), plotted over the pixel shift ξ (RICS curves, fixed τ p ) and the pixel dwell time τ p /line time τ l (msICS curves, fixed ξ).

Fig. 6
Fig. 6

Map of msICS-derived diffusion coefficients of a HeLa cell expressing EGFP. (a) A 10.5×12.0 μm2 area covering parts of the cytoplasm, the nucleus and the nucleoli was sampled with 13×15 ROIs (50×50 pixels, 1.5×1.5 μm2, white squares) that were centered around the white crosses corresponding to two-fold oversampling and used for msICS analysis (scale bar 5 μm). (b) autocorrelation curves ( ξ = 1 , circles) and fits (red lines) of Eq. (2) acquired at positions in a nucleolus, the nucleus and the cytoplasm yielding diffusion coefficients of 8.1, 7.6, and 5.6 μm2 s–1, respectively. (c) Resulting contour map of diffusion coefficients (black for areas where the correlation analysis failed – n/d, not determined).

Tables (2)

Tables Icon

Table 1 Diffusion coefficients D from RICS and FCS experiments in EGFP-expressing HeLa cells

Tables Icon

Table 2 Diffusion coefficients D from msICS experiments of Alexa488

Equations (8)

Equations on this page are rendered with MathJax. Learn more.

G RICS ( ξ , φ ) = I ( x , y ) I ( x + ξ , y + φ ) I ( x , y ) 2 .
G RICS = 1 N [ 1 + 4 D ( τ p ξ + τ l φ ) w 0 2 ] 1 [ 1 + 4 D ( τ p ξ + τ l φ ) κ 2 w 0 2 ] 1 / 2 = G D × exp [ ( δ x ξ / w 0 ) 2 + ( δ y φ / w 0 ) 2 1 + 4 D ( τ p ξ + τ l φ ) / w 0 2 ] = G S × [ 1 + θ exp ( τ p ξ + τ l φ τ trip ) ]
P ( r , t ) = ( 4 π D t ) 3 / 2 exp ( r 2 4 D t ) .
P t | t = t 0 = 0 t 0 = r 0 2 6 D .
M corr,i new = I ¯ i I ¯ 0 M corr,i old
v p,i new = v p,i old ( v ¯ local , d v ¯ global ) .
F I I + I s
G ( τ ) = 1 N ( f 1 [ 1 + ( τ τ diff , 1 ) α 1 ] 1 [ 1 + 1 κ 2 ( τ τ diff , 1 ) α 1 ] 1 / 2 + ( 1 f 1 ) [ 1 + ( τ τ diff , 2 ) α 2 ] 1 [ 1 + 1 κ 2 ( τ τ diff , 2 ) α 2 ] 1 / 2 ) [ 1 + θ exp ( τ τ trip ) ]

Metrics