Abstract

We characterize a novel fluorescence microscope which combines the high spatial discrimination of a total internal reflection epi-fluorescence (epi-TIRF) microscope with that of stimulated emission depletion (STED) nanoscopy. This combination of high axial confinement and dynamic-active lateral spatial discrimination of the detected fluorescence emission promises imaging and spectroscopy of the structure and function of cell membranes at the macro-molecular scale. Following a full theoretical description of the sampling volume and the recording of images of fluorescent beads, we exemplify the performance and limitations of the TIRF-STED nanoscope with particular attention to the polarization state of the laser excitation light. We demonstrate fluorescence correlation spectroscopy (FCS) with the TIRF-STED nanoscope by observing the diffusion of dye molecules in aqueous solutions and of fluorescent lipid analogs in supported lipid bilayers in the presence of background signal. The nanoscope reduced the out-of-focus background signal. A lateral resolution down to 40–50 nm was attained which was ultimately limited by the low lateral signal-to-background ratio inherent to the confocal epi-TIRF scheme. Together with the estimated axial confinement of about 55 nm, our TIRF-STED nanoscope achieved an almost isotropic and less than 1 attoliter small all-optically induced measurement volume.

© 2012 OSA

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. J. B. Pawley, Handbook of Biological Confocal Microscopy, 3rd ed. (Springer, New York 2005).
  2. E. Abbe, “Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung,” Archiv für Mikroskopische Anatomie 9(1), 413–418 (1873).
    [CrossRef]
  3. S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 780–782 (1994).
    [CrossRef] [PubMed]
  4. T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
    [CrossRef] [PubMed]
  5. S. W. Hell, “Far-field optical nanoscopy,” Science 316(5828), 1153–1158 (2007).
    [CrossRef] [PubMed]
  6. V. Westphal and S. W. Hell, “Nanoscale resolution in the focal plane of an optical microscope,” Phys. Rev. Lett. 94(14), 143903 (2005).
    [CrossRef] [PubMed]
  7. B. Harke, C. K. Ullal, J. Keller, and S. W. Hell, “Three-dimensional nanoscopy of colloidal crystals,” Nano Lett. 8(5), 1309–1313 (2008).
    [CrossRef] [PubMed]
  8. D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. 236(1), 35–43 (2009).
    [CrossRef] [PubMed]
  9. R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
    [CrossRef] [PubMed]
  10. D. Magde, W. W. Webb, and E. Elson, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
    [CrossRef]
  11. M. Ehrenberg and R. Rigler, “Fluorescence correlation spectroscopy applied to rotational diffusion of macromolecules,” Q. Rev. Biophys. 9(1), 69–81 (1976).
    [CrossRef] [PubMed]
  12. C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
    [CrossRef] [PubMed]
  13. L. Kastrup, H. Blom, C. Eggeling, and S. W. Hell, “Fluorescence fluctuation spectroscopy in subdiffraction focal volumes,” Phys. Rev. Lett. 94(17), 178104 (2005).
    [CrossRef] [PubMed]
  14. C. Ringemann, B. Harke, C. von Middendorff, R. Medda, A. Honigmann, R. Wagner, M. Leutenegger, A. Schönle, S. W. Hell, and C. Eggeling, “Exploring single-molecule dynamics with fluorescence nanoscopy,” New J. Phys. 11(10), 103054 (2009).
    [CrossRef]
  15. D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol. 89(1), 141–145 (1981).
    [CrossRef] [PubMed]
  16. G. A. Truskey, J. S. Burmeister, E. Grapa, and W. M. Reichert, “Total internal reflection fluorescence microscopy (TIRFM). II. Topographical mapping of relative cell/substratum separation distances,” J. Cell Sci. 103(Pt 2), 491–499 (1992).
    [PubMed]
  17. M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, and T. Yanagida, “Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy,” Biochem. Biophys. Res. Commun. 235(1), 47–53 (1997).
    [CrossRef] [PubMed]
  18. A. B. Mathur, G. A. Truskey, and W. M. Reichert, “Atomic force and total internal reflection fluorescence microscopy for the study of force transmission in endothelial cells,” Biophys. J. 78(4), 1725–1735 (2000).
    [CrossRef] [PubMed]
  19. D. Axelrod, “Total internal reflection fluorescence microscopy in cell biology,” Traffic 2(11), 764–774 (2001).
    [CrossRef] [PubMed]
  20. H. Schneckenburger, “Total internal reflection fluorescence microscopy: technical innovations and novel applications,” Curr. Opin. Biotechnol. 16(1), 13–18 (2005).
    [CrossRef] [PubMed]
  21. A. M. Lieto, R. C. Cush, and N. L. Thompson, “Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy,” Biophys. J. 85(5), 3294–3302 (2003).
    [CrossRef] [PubMed]
  22. D. Axelrod, E. H. Hellen, and R. M. Fulbright, “Total internal reflection fluorescence” in Topics in Fluorescence Spectroscopy, J. R. Lakowicz (ed.) (Springer 2002), Vol. 3, pp. 289–343.
  23. T. Ruckstuhl and D. Verdes, “Supercritical angle fluorescence (SAF) microscopy,” Opt. Express 12(18), 4246–4254 (2004).
    [CrossRef] [PubMed]
  24. N. L. Thompson, T. P. Burghardt, and D. Axelrod, “Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy,” Biophys. J. 33(3), 435–454 (1981).
    [CrossRef] [PubMed]
  25. K. Hassler, T. Anhut, R. Rigler, M. Gösch, and T. Lasser, “High count rates with total internal reflection fluorescence correlation spectroscopy,” Biophys. J. 88(1), L01–L03 (2005).
    [CrossRef] [PubMed]
  26. W. T. Welford, “Use of annular apertures to increase focal depth,” J. Opt. Soc. Am. 50(8), 749–753 (1960).
    [CrossRef]
  27. R. M. Herman and T. A. Wiggins, “Production and uses of diffractionless beams,” J. Opt. Soc. Am. A 8(6), 932–942 (1991).
    [CrossRef]
  28. T. Ruckstuhl and S. Seeger, “Attoliter detection volumes by confocal total-internal-reflection fluorescence microscopy,” Opt. Lett. 29(6), 569–571 (2004).
    [CrossRef] [PubMed]
  29. J. W. M. Chon, M. Gu, C. Bullen, and P. Mulvaney, “Two-photon fluorescence scanning near-field microscopy based on a focused evanescent field under total internal reflection,” Opt. Lett. 28(20), 1930–1932 (2003).
    [CrossRef] [PubMed]
  30. T. J. Gould, J. R. Myers, and J. Bewersdorf, “Total internal reflection STED microscopy,” Opt. Express 19(14), 13351–13357 (2011).
    [CrossRef] [PubMed]
  31. K. I. Willig, J. Keller, M. Bossi, and S. W. Hell, “STED microscopy resolves nanoparticle assemblies,” New J. Phys. 8(6), 106 (2006).
    [CrossRef]
  32. M. Leutenegger, R. Rao, R. A. Leitgeb, and T. Lasser, “Fast focus field calculations,” Opt. Express 14(23), 11277–11291 (2006).
    [CrossRef] [PubMed]
  33. M. Leutenegger and T. Lasser, “Detection efficiency in total internal reflection fluorescence microscopy,” Opt. Express 16(12), 8519–8531 (2008).
    [CrossRef] [PubMed]
  34. R. Dorn, S. Quabis, and G. Leuchs, “Sharper focus for a radially polarized light beam,” Phys. Rev. Lett. 91(23), 233901 (2003).
    [CrossRef] [PubMed]
  35. M. Leutenegger, C. Eggeling, and S. W. Hell, “Analytical description of STED microscopy performance,” Opt. Express 18(25), 26417–26429 (2010).
    [CrossRef] [PubMed]
  36. W. Lukosz and R. E. Kunz, “Light-emission by magnetic and electric dipoles close to a plane interface: 1. Total radiated power,” J. Opt. Soc. Am. 67(12), 1607–1615 (1977).
    [CrossRef]
  37. G. W. Ford and W. H. Weber, “Electromagnetic interactions of molecules with metal surfaces,” Phys. Rep. 113(4), 195–287 (1984).
    [CrossRef]
  38. A. Honigmann, C. Walter, F. Erdmann, C. Eggeling, and R. Wagner, “Characterization of horizontal lipid bilayers as a model system to study lipid phase separation,” Biophys. J. 98(12), 2886–2894 (2010).
    [CrossRef] [PubMed]
  39. S. Chiantia, N. Kahya, and P. Schwille, “Dehydration damage of domain-exhibiting supported bilayers: an AFM study on the protective effects of disaccharides and other stabilizing substances,” Langmuir 21(14), 6317–6323 (2005).
    [CrossRef] [PubMed]
  40. J. Widengren, U. 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]
  41. S. Chiantia, J. Ries, N. Kahya, and P. Schwille, “Combined AFM and two-focus SFCS study of raft-exhibiting model membranes,” ChemPhysChem 7(11), 2409–2418 (2006).
    [CrossRef] [PubMed]
  42. U. Golebiewska, M. Nyako, W. Woturski, I. Zaitseva, and S. McLaughlin, “Diffusion coefficient of fluorescent phosphatidylinositol 4,5-bisphosphate in the plasma membrane of cells,” Mol. Biol. Cell 19(4), 1663–1669 (2008).
    [CrossRef] [PubMed]
  43. T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
    [CrossRef] [PubMed]
  44. D. E. Koppel, “Statistical accuracy in fluorescence correlation spectroscopy,” Phys. Rev. A 10(6), 1938–1945 (1974).
    [CrossRef]
  45. E. Rittweger, B. R. Rankin, V. Westphal, and S. W. Hell, “Fluorescence depletion mechanisms in super-resolving STED microscopy,” Chem. Phys. Lett. 442(4-6), 483–487 (2007).
    [CrossRef]
  46. K. Kolmakov, V. N. Belov, J. Bierwagen, C. Ringemann, V. Müller, C. Eggeling, and S. W. Hell, “Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy,” Chemistry 16(1), 158–166 (2010).
    [CrossRef] [PubMed]
  47. S. W. Hell and E. H. K. Stelzer, “Properties of a 4Pi confocal fluorescence microscope,” J. Opt. Soc. Am. A 9(12), 2159–2166 (1992).
    [CrossRef]
  48. M. Gu and C. J. R. Sheppard, “Three-dimensional transfer functions in 4Pi confocal microscopes,” J. Opt. Soc. Am. A 11(5), 1619–1627 (1994).
    [CrossRef]
  49. K. Hassler, Single molecule detection and fluorescence correlation spectroscopy on surfaces, doctoral thesis, École Polytechnique Fédérale de Lausanne, Switzerland (2005): http://library.epfl.ch/theses/?nr=3433 .

2011

2010

M. Leutenegger, C. Eggeling, and S. W. Hell, “Analytical description of STED microscopy performance,” Opt. Express 18(25), 26417–26429 (2010).
[CrossRef] [PubMed]

A. Honigmann, C. Walter, F. Erdmann, C. Eggeling, and R. Wagner, “Characterization of horizontal lipid bilayers as a model system to study lipid phase separation,” Biophys. J. 98(12), 2886–2894 (2010).
[CrossRef] [PubMed]

K. Kolmakov, V. N. Belov, J. Bierwagen, C. Ringemann, V. Müller, C. Eggeling, and S. W. Hell, “Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy,” Chemistry 16(1), 158–166 (2010).
[CrossRef] [PubMed]

2009

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. 236(1), 35–43 (2009).
[CrossRef] [PubMed]

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

C. Ringemann, B. Harke, C. von Middendorff, R. Medda, A. Honigmann, R. Wagner, M. Leutenegger, A. Schönle, S. W. Hell, and C. Eggeling, “Exploring single-molecule dynamics with fluorescence nanoscopy,” New J. Phys. 11(10), 103054 (2009).
[CrossRef]

2008

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[CrossRef] [PubMed]

B. Harke, C. K. Ullal, J. Keller, and S. W. Hell, “Three-dimensional nanoscopy of colloidal crystals,” Nano Lett. 8(5), 1309–1313 (2008).
[CrossRef] [PubMed]

M. Leutenegger and T. Lasser, “Detection efficiency in total internal reflection fluorescence microscopy,” Opt. Express 16(12), 8519–8531 (2008).
[CrossRef] [PubMed]

U. Golebiewska, M. Nyako, W. Woturski, I. Zaitseva, and S. McLaughlin, “Diffusion coefficient of fluorescent phosphatidylinositol 4,5-bisphosphate in the plasma membrane of cells,” Mol. Biol. Cell 19(4), 1663–1669 (2008).
[CrossRef] [PubMed]

2007

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[CrossRef] [PubMed]

E. Rittweger, B. R. Rankin, V. Westphal, and S. W. Hell, “Fluorescence depletion mechanisms in super-resolving STED microscopy,” Chem. Phys. Lett. 442(4-6), 483–487 (2007).
[CrossRef]

S. W. Hell, “Far-field optical nanoscopy,” Science 316(5828), 1153–1158 (2007).
[CrossRef] [PubMed]

2006

K. I. Willig, J. Keller, M. Bossi, and S. W. Hell, “STED microscopy resolves nanoparticle assemblies,” New J. Phys. 8(6), 106 (2006).
[CrossRef]

M. Leutenegger, R. Rao, R. A. Leitgeb, and T. Lasser, “Fast focus field calculations,” Opt. Express 14(23), 11277–11291 (2006).
[CrossRef] [PubMed]

S. Chiantia, J. Ries, N. Kahya, and P. Schwille, “Combined AFM and two-focus SFCS study of raft-exhibiting model membranes,” ChemPhysChem 7(11), 2409–2418 (2006).
[CrossRef] [PubMed]

2005

S. Chiantia, N. Kahya, and P. Schwille, “Dehydration damage of domain-exhibiting supported bilayers: an AFM study on the protective effects of disaccharides and other stabilizing substances,” Langmuir 21(14), 6317–6323 (2005).
[CrossRef] [PubMed]

H. Schneckenburger, “Total internal reflection fluorescence microscopy: technical innovations and novel applications,” Curr. Opin. Biotechnol. 16(1), 13–18 (2005).
[CrossRef] [PubMed]

K. Hassler, T. Anhut, R. Rigler, M. Gösch, and T. Lasser, “High count rates with total internal reflection fluorescence correlation spectroscopy,” Biophys. J. 88(1), L01–L03 (2005).
[CrossRef] [PubMed]

V. Westphal and S. W. Hell, “Nanoscale resolution in the focal plane of an optical microscope,” Phys. Rev. Lett. 94(14), 143903 (2005).
[CrossRef] [PubMed]

L. Kastrup, H. Blom, C. Eggeling, and S. W. Hell, “Fluorescence fluctuation spectroscopy in subdiffraction focal volumes,” Phys. Rev. Lett. 94(17), 178104 (2005).
[CrossRef] [PubMed]

2004

2003

J. W. M. Chon, M. Gu, C. Bullen, and P. Mulvaney, “Two-photon fluorescence scanning near-field microscopy based on a focused evanescent field under total internal reflection,” Opt. Lett. 28(20), 1930–1932 (2003).
[CrossRef] [PubMed]

R. Dorn, S. Quabis, and G. Leuchs, “Sharper focus for a radially polarized light beam,” Phys. Rev. Lett. 91(23), 233901 (2003).
[CrossRef] [PubMed]

A. M. Lieto, R. C. Cush, and N. L. Thompson, “Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy,” Biophys. J. 85(5), 3294–3302 (2003).
[CrossRef] [PubMed]

2001

D. Axelrod, “Total internal reflection fluorescence microscopy in cell biology,” Traffic 2(11), 764–774 (2001).
[CrossRef] [PubMed]

2000

A. B. Mathur, G. A. Truskey, and W. M. Reichert, “Atomic force and total internal reflection fluorescence microscopy for the study of force transmission in endothelial cells,” Biophys. J. 78(4), 1725–1735 (2000).
[CrossRef] [PubMed]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

1997

M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, and T. Yanagida, “Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy,” Biochem. Biophys. Res. Commun. 235(1), 47–53 (1997).
[CrossRef] [PubMed]

1995

J. Widengren, U. 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

1992

G. A. Truskey, J. S. Burmeister, E. Grapa, and W. M. Reichert, “Total internal reflection fluorescence microscopy (TIRFM). II. Topographical mapping of relative cell/substratum separation distances,” J. Cell Sci. 103(Pt 2), 491–499 (1992).
[PubMed]

S. W. Hell and E. H. K. Stelzer, “Properties of a 4Pi confocal fluorescence microscope,” J. Opt. Soc. Am. A 9(12), 2159–2166 (1992).
[CrossRef]

1991

1984

G. W. Ford and W. H. Weber, “Electromagnetic interactions of molecules with metal surfaces,” Phys. Rep. 113(4), 195–287 (1984).
[CrossRef]

1981

N. L. Thompson, T. P. Burghardt, and D. Axelrod, “Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy,” Biophys. J. 33(3), 435–454 (1981).
[CrossRef] [PubMed]

D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol. 89(1), 141–145 (1981).
[CrossRef] [PubMed]

1977

1976

M. Ehrenberg and R. Rigler, “Fluorescence correlation spectroscopy applied to rotational diffusion of macromolecules,” Q. Rev. Biophys. 9(1), 69–81 (1976).
[CrossRef] [PubMed]

1974

D. E. Koppel, “Statistical accuracy in fluorescence correlation spectroscopy,” Phys. Rev. A 10(6), 1938–1945 (1974).
[CrossRef]

1972

D. Magde, W. W. Webb, and E. Elson, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[CrossRef]

1960

1873

E. Abbe, “Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung,” Archiv für Mikroskopische Anatomie 9(1), 413–418 (1873).
[CrossRef]

Abbe, E.

E. Abbe, “Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung,” Archiv für Mikroskopische Anatomie 9(1), 413–418 (1873).
[CrossRef]

Anhut, T.

K. Hassler, T. Anhut, R. Rigler, M. Gösch, and T. Lasser, “High count rates with total internal reflection fluorescence correlation spectroscopy,” Biophys. J. 88(1), L01–L03 (2005).
[CrossRef] [PubMed]

Axelrod, D.

D. Axelrod, “Total internal reflection fluorescence microscopy in cell biology,” Traffic 2(11), 764–774 (2001).
[CrossRef] [PubMed]

D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol. 89(1), 141–145 (1981).
[CrossRef] [PubMed]

N. L. Thompson, T. P. Burghardt, and D. Axelrod, “Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy,” Biophys. J. 33(3), 435–454 (1981).
[CrossRef] [PubMed]

Belov, V. N.

K. Kolmakov, V. N. Belov, J. Bierwagen, C. Ringemann, V. Müller, C. Eggeling, and S. W. Hell, “Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy,” Chemistry 16(1), 158–166 (2010).
[CrossRef] [PubMed]

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Bewersdorf, J.

Bierwagen, J.

K. Kolmakov, V. N. Belov, J. Bierwagen, C. Ringemann, V. Müller, C. Eggeling, and S. W. Hell, “Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy,” Chemistry 16(1), 158–166 (2010).
[CrossRef] [PubMed]

Blom, H.

L. Kastrup, H. Blom, C. Eggeling, and S. W. Hell, “Fluorescence fluctuation spectroscopy in subdiffraction focal volumes,” Phys. Rev. Lett. 94(17), 178104 (2005).
[CrossRef] [PubMed]

Bossi, M.

K. I. Willig, J. Keller, M. Bossi, and S. W. Hell, “STED microscopy resolves nanoparticle assemblies,” New J. Phys. 8(6), 106 (2006).
[CrossRef]

Bullen, C.

Burghardt, T. P.

N. L. Thompson, T. P. Burghardt, and D. Axelrod, “Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy,” Biophys. J. 33(3), 435–454 (1981).
[CrossRef] [PubMed]

Burmeister, J. S.

G. A. Truskey, J. S. Burmeister, E. Grapa, and W. M. Reichert, “Total internal reflection fluorescence microscopy (TIRFM). II. Topographical mapping of relative cell/substratum separation distances,” J. Cell Sci. 103(Pt 2), 491–499 (1992).
[PubMed]

Chiantia, S.

S. Chiantia, J. Ries, N. Kahya, and P. Schwille, “Combined AFM and two-focus SFCS study of raft-exhibiting model membranes,” ChemPhysChem 7(11), 2409–2418 (2006).
[CrossRef] [PubMed]

S. Chiantia, N. Kahya, and P. Schwille, “Dehydration damage of domain-exhibiting supported bilayers: an AFM study on the protective effects of disaccharides and other stabilizing substances,” Langmuir 21(14), 6317–6323 (2005).
[CrossRef] [PubMed]

Chon, J. W. M.

Cush, R. C.

A. M. Lieto, R. C. Cush, and N. L. Thompson, “Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy,” Biophys. J. 85(5), 3294–3302 (2003).
[CrossRef] [PubMed]

Dertinger, T.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[CrossRef] [PubMed]

Dorn, R.

R. Dorn, S. Quabis, and G. Leuchs, “Sharper focus for a radially polarized light beam,” Phys. Rev. Lett. 91(23), 233901 (2003).
[CrossRef] [PubMed]

Dyba, M.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

Eggeling, C.

A. Honigmann, C. Walter, F. Erdmann, C. Eggeling, and R. Wagner, “Characterization of horizontal lipid bilayers as a model system to study lipid phase separation,” Biophys. J. 98(12), 2886–2894 (2010).
[CrossRef] [PubMed]

M. Leutenegger, C. Eggeling, and S. W. Hell, “Analytical description of STED microscopy performance,” Opt. Express 18(25), 26417–26429 (2010).
[CrossRef] [PubMed]

K. Kolmakov, V. N. Belov, J. Bierwagen, C. Ringemann, V. Müller, C. Eggeling, and S. W. Hell, “Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy,” Chemistry 16(1), 158–166 (2010).
[CrossRef] [PubMed]

C. Ringemann, B. Harke, C. von Middendorff, R. Medda, A. Honigmann, R. Wagner, M. Leutenegger, A. Schönle, S. W. Hell, and C. Eggeling, “Exploring single-molecule dynamics with fluorescence nanoscopy,” New J. Phys. 11(10), 103054 (2009).
[CrossRef]

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

L. Kastrup, H. Blom, C. Eggeling, and S. W. Hell, “Fluorescence fluctuation spectroscopy in subdiffraction focal volumes,” Phys. Rev. Lett. 94(17), 178104 (2005).
[CrossRef] [PubMed]

Egner, A.

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[CrossRef] [PubMed]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

Ehrenberg, M.

M. Ehrenberg and R. Rigler, “Fluorescence correlation spectroscopy applied to rotational diffusion of macromolecules,” Q. Rev. Biophys. 9(1), 69–81 (1976).
[CrossRef] [PubMed]

Elson, E.

D. Magde, W. W. Webb, and E. Elson, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[CrossRef]

Enderlein, J.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[CrossRef] [PubMed]

Engelhardt, J.

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[CrossRef] [PubMed]

Erdmann, F.

A. Honigmann, C. Walter, F. Erdmann, C. Eggeling, and R. Wagner, “Characterization of horizontal lipid bilayers as a model system to study lipid phase separation,” Biophys. J. 98(12), 2886–2894 (2010).
[CrossRef] [PubMed]

Ford, G. W.

G. W. Ford and W. H. Weber, “Electromagnetic interactions of molecules with metal surfaces,” Phys. Rep. 113(4), 195–287 (1984).
[CrossRef]

Golebiewska, U.

U. Golebiewska, M. Nyako, W. Woturski, I. Zaitseva, and S. McLaughlin, “Diffusion coefficient of fluorescent phosphatidylinositol 4,5-bisphosphate in the plasma membrane of cells,” Mol. Biol. Cell 19(4), 1663–1669 (2008).
[CrossRef] [PubMed]

Gösch, M.

K. Hassler, T. Anhut, R. Rigler, M. Gösch, and T. Lasser, “High count rates with total internal reflection fluorescence correlation spectroscopy,” Biophys. J. 88(1), L01–L03 (2005).
[CrossRef] [PubMed]

Gould, T. J.

Grapa, E.

G. A. Truskey, J. S. Burmeister, E. Grapa, and W. M. Reichert, “Total internal reflection fluorescence microscopy (TIRFM). II. Topographical mapping of relative cell/substratum separation distances,” J. Cell Sci. 103(Pt 2), 491–499 (1992).
[PubMed]

Gregor, I.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[CrossRef] [PubMed]

Gu, M.

Harke, B.

C. Ringemann, B. Harke, C. von Middendorff, R. Medda, A. Honigmann, R. Wagner, M. Leutenegger, A. Schönle, S. W. Hell, and C. Eggeling, “Exploring single-molecule dynamics with fluorescence nanoscopy,” New J. Phys. 11(10), 103054 (2009).
[CrossRef]

B. Harke, C. K. Ullal, J. Keller, and S. W. Hell, “Three-dimensional nanoscopy of colloidal crystals,” Nano Lett. 8(5), 1309–1313 (2008).
[CrossRef] [PubMed]

Hartmann, R.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[CrossRef] [PubMed]

Hassler, K.

K. Hassler, T. Anhut, R. Rigler, M. Gösch, and T. Lasser, “High count rates with total internal reflection fluorescence correlation spectroscopy,” Biophys. J. 88(1), L01–L03 (2005).
[CrossRef] [PubMed]

Hein, B.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Hell, S. W.

K. Kolmakov, V. N. Belov, J. Bierwagen, C. Ringemann, V. Müller, C. Eggeling, and S. W. Hell, “Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy,” Chemistry 16(1), 158–166 (2010).
[CrossRef] [PubMed]

M. Leutenegger, C. Eggeling, and S. W. Hell, “Analytical description of STED microscopy performance,” Opt. Express 18(25), 26417–26429 (2010).
[CrossRef] [PubMed]

C. Ringemann, B. Harke, C. von Middendorff, R. Medda, A. Honigmann, R. Wagner, M. Leutenegger, A. Schönle, S. W. Hell, and C. Eggeling, “Exploring single-molecule dynamics with fluorescence nanoscopy,” New J. Phys. 11(10), 103054 (2009).
[CrossRef]

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. 236(1), 35–43 (2009).
[CrossRef] [PubMed]

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[CrossRef] [PubMed]

B. Harke, C. K. Ullal, J. Keller, and S. W. Hell, “Three-dimensional nanoscopy of colloidal crystals,” Nano Lett. 8(5), 1309–1313 (2008).
[CrossRef] [PubMed]

S. W. Hell, “Far-field optical nanoscopy,” Science 316(5828), 1153–1158 (2007).
[CrossRef] [PubMed]

E. Rittweger, B. R. Rankin, V. Westphal, and S. W. Hell, “Fluorescence depletion mechanisms in super-resolving STED microscopy,” Chem. Phys. Lett. 442(4-6), 483–487 (2007).
[CrossRef]

K. I. Willig, J. Keller, M. Bossi, and S. W. Hell, “STED microscopy resolves nanoparticle assemblies,” New J. Phys. 8(6), 106 (2006).
[CrossRef]

V. Westphal and S. W. Hell, “Nanoscale resolution in the focal plane of an optical microscope,” Phys. Rev. Lett. 94(14), 143903 (2005).
[CrossRef] [PubMed]

L. Kastrup, H. Blom, C. Eggeling, and S. W. Hell, “Fluorescence fluctuation spectroscopy in subdiffraction focal volumes,” Phys. Rev. Lett. 94(17), 178104 (2005).
[CrossRef] [PubMed]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 780–782 (1994).
[CrossRef] [PubMed]

S. W. Hell and E. H. K. Stelzer, “Properties of a 4Pi confocal fluorescence microscope,” J. Opt. Soc. Am. A 9(12), 2159–2166 (1992).
[CrossRef]

Herman, R. M.

Honigmann, A.

A. Honigmann, C. Walter, F. Erdmann, C. Eggeling, and R. Wagner, “Characterization of horizontal lipid bilayers as a model system to study lipid phase separation,” Biophys. J. 98(12), 2886–2894 (2010).
[CrossRef] [PubMed]

C. Ringemann, B. Harke, C. von Middendorff, R. Medda, A. Honigmann, R. Wagner, M. Leutenegger, A. Schönle, S. W. Hell, and C. Eggeling, “Exploring single-molecule dynamics with fluorescence nanoscopy,” New J. Phys. 11(10), 103054 (2009).
[CrossRef]

Iwane, A. H.

M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, and T. Yanagida, “Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy,” Biochem. Biophys. Res. Commun. 235(1), 47–53 (1997).
[CrossRef] [PubMed]

Jakobs, S.

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[CrossRef] [PubMed]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

Kahya, N.

S. Chiantia, J. Ries, N. Kahya, and P. Schwille, “Combined AFM and two-focus SFCS study of raft-exhibiting model membranes,” ChemPhysChem 7(11), 2409–2418 (2006).
[CrossRef] [PubMed]

S. Chiantia, N. Kahya, and P. Schwille, “Dehydration damage of domain-exhibiting supported bilayers: an AFM study on the protective effects of disaccharides and other stabilizing substances,” Langmuir 21(14), 6317–6323 (2005).
[CrossRef] [PubMed]

Kastrup, L.

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. 236(1), 35–43 (2009).
[CrossRef] [PubMed]

L. Kastrup, H. Blom, C. Eggeling, and S. W. Hell, “Fluorescence fluctuation spectroscopy in subdiffraction focal volumes,” Phys. Rev. Lett. 94(17), 178104 (2005).
[CrossRef] [PubMed]

Keller, J.

B. Harke, C. K. Ullal, J. Keller, and S. W. Hell, “Three-dimensional nanoscopy of colloidal crystals,” Nano Lett. 8(5), 1309–1313 (2008).
[CrossRef] [PubMed]

K. I. Willig, J. Keller, M. Bossi, and S. W. Hell, “STED microscopy resolves nanoparticle assemblies,” New J. Phys. 8(6), 106 (2006).
[CrossRef]

Kitamura, K.

M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, and T. Yanagida, “Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy,” Biochem. Biophys. Res. Commun. 235(1), 47–53 (1997).
[CrossRef] [PubMed]

Klar, T. A.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

Kolmakov, K.

K. Kolmakov, V. N. Belov, J. Bierwagen, C. Ringemann, V. Müller, C. Eggeling, and S. W. Hell, “Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy,” Chemistry 16(1), 158–166 (2010).
[CrossRef] [PubMed]

Koppel, D. E.

D. E. Koppel, “Statistical accuracy in fluorescence correlation spectroscopy,” Phys. Rev. A 10(6), 1938–1945 (1974).
[CrossRef]

Kunz, R. E.

Lasser, T.

Leitgeb, R. A.

Leuchs, G.

R. Dorn, S. Quabis, and G. Leuchs, “Sharper focus for a radially polarized light beam,” Phys. Rev. Lett. 91(23), 233901 (2003).
[CrossRef] [PubMed]

Leutenegger, M.

Lieto, A. M.

A. M. Lieto, R. C. Cush, and N. L. Thompson, “Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy,” Biophys. J. 85(5), 3294–3302 (2003).
[CrossRef] [PubMed]

Lukosz, W.

Magde, D.

D. Magde, W. W. Webb, and E. Elson, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[CrossRef]

Mathur, A. B.

A. B. Mathur, G. A. Truskey, and W. M. Reichert, “Atomic force and total internal reflection fluorescence microscopy for the study of force transmission in endothelial cells,” Biophys. J. 78(4), 1725–1735 (2000).
[CrossRef] [PubMed]

McLaughlin, S.

U. Golebiewska, M. Nyako, W. Woturski, I. Zaitseva, and S. McLaughlin, “Diffusion coefficient of fluorescent phosphatidylinositol 4,5-bisphosphate in the plasma membrane of cells,” Mol. Biol. Cell 19(4), 1663–1669 (2008).
[CrossRef] [PubMed]

Medda, R.

C. Ringemann, B. Harke, C. von Middendorff, R. Medda, A. Honigmann, R. Wagner, M. Leutenegger, A. Schönle, S. W. Hell, and C. Eggeling, “Exploring single-molecule dynamics with fluorescence nanoscopy,” New J. Phys. 11(10), 103054 (2009).
[CrossRef]

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. 236(1), 35–43 (2009).
[CrossRef] [PubMed]

Mets, U.

J. Widengren, U. 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]

Müller, V.

K. Kolmakov, V. N. Belov, J. Bierwagen, C. Ringemann, V. Müller, C. Eggeling, and S. W. Hell, “Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy,” Chemistry 16(1), 158–166 (2010).
[CrossRef] [PubMed]

Mulvaney, P.

Myers, J. R.

Nyako, M.

U. Golebiewska, M. Nyako, W. Woturski, I. Zaitseva, and S. McLaughlin, “Diffusion coefficient of fluorescent phosphatidylinositol 4,5-bisphosphate in the plasma membrane of cells,” Mol. Biol. Cell 19(4), 1663–1669 (2008).
[CrossRef] [PubMed]

Pacheco, V.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[CrossRef] [PubMed]

Polyakova, S.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Quabis, S.

R. Dorn, S. Quabis, and G. Leuchs, “Sharper focus for a radially polarized light beam,” Phys. Rev. Lett. 91(23), 233901 (2003).
[CrossRef] [PubMed]

Rankin, B. R.

E. Rittweger, B. R. Rankin, V. Westphal, and S. W. Hell, “Fluorescence depletion mechanisms in super-resolving STED microscopy,” Chem. Phys. Lett. 442(4-6), 483–487 (2007).
[CrossRef]

Rao, R.

Reichert, W. M.

A. B. Mathur, G. A. Truskey, and W. M. Reichert, “Atomic force and total internal reflection fluorescence microscopy for the study of force transmission in endothelial cells,” Biophys. J. 78(4), 1725–1735 (2000).
[CrossRef] [PubMed]

G. A. Truskey, J. S. Burmeister, E. Grapa, and W. M. Reichert, “Total internal reflection fluorescence microscopy (TIRFM). II. Topographical mapping of relative cell/substratum separation distances,” J. Cell Sci. 103(Pt 2), 491–499 (1992).
[PubMed]

Ries, J.

S. Chiantia, J. Ries, N. Kahya, and P. Schwille, “Combined AFM and two-focus SFCS study of raft-exhibiting model membranes,” ChemPhysChem 7(11), 2409–2418 (2006).
[CrossRef] [PubMed]

Rigler, R.

K. Hassler, T. Anhut, R. Rigler, M. Gösch, and T. Lasser, “High count rates with total internal reflection fluorescence correlation spectroscopy,” Biophys. J. 88(1), L01–L03 (2005).
[CrossRef] [PubMed]

J. Widengren, U. 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]

M. Ehrenberg and R. Rigler, “Fluorescence correlation spectroscopy applied to rotational diffusion of macromolecules,” Q. Rev. Biophys. 9(1), 69–81 (1976).
[CrossRef] [PubMed]

Ringemann, C.

K. Kolmakov, V. N. Belov, J. Bierwagen, C. Ringemann, V. Müller, C. Eggeling, and S. W. Hell, “Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy,” Chemistry 16(1), 158–166 (2010).
[CrossRef] [PubMed]

C. Ringemann, B. Harke, C. von Middendorff, R. Medda, A. Honigmann, R. Wagner, M. Leutenegger, A. Schönle, S. W. Hell, and C. Eggeling, “Exploring single-molecule dynamics with fluorescence nanoscopy,” New J. Phys. 11(10), 103054 (2009).
[CrossRef]

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Rittweger, E.

E. Rittweger, B. R. Rankin, V. Westphal, and S. W. Hell, “Fluorescence depletion mechanisms in super-resolving STED microscopy,” Chem. Phys. Lett. 442(4-6), 483–487 (2007).
[CrossRef]

Ruckstuhl, T.

Saito, K.

M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, and T. Yanagida, “Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy,” Biochem. Biophys. Res. Commun. 235(1), 47–53 (1997).
[CrossRef] [PubMed]

Sandhoff, K.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Schmidt, R.

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[CrossRef] [PubMed]

Schneckenburger, H.

H. Schneckenburger, “Total internal reflection fluorescence microscopy: technical innovations and novel applications,” Curr. Opin. Biotechnol. 16(1), 13–18 (2005).
[CrossRef] [PubMed]

Schönle, A.

C. Ringemann, B. Harke, C. von Middendorff, R. Medda, A. Honigmann, R. Wagner, M. Leutenegger, A. Schönle, S. W. Hell, and C. Eggeling, “Exploring single-molecule dynamics with fluorescence nanoscopy,” New J. Phys. 11(10), 103054 (2009).
[CrossRef]

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Schwarzmann, G.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Schwille, P.

S. Chiantia, J. Ries, N. Kahya, and P. Schwille, “Combined AFM and two-focus SFCS study of raft-exhibiting model membranes,” ChemPhysChem 7(11), 2409–2418 (2006).
[CrossRef] [PubMed]

S. Chiantia, N. Kahya, and P. Schwille, “Dehydration damage of domain-exhibiting supported bilayers: an AFM study on the protective effects of disaccharides and other stabilizing substances,” Langmuir 21(14), 6317–6323 (2005).
[CrossRef] [PubMed]

Seeger, S.

Sheppard, C. J. R.

Stelzer, E. H. K.

Thompson, N. L.

A. M. Lieto, R. C. Cush, and N. L. Thompson, “Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy,” Biophys. J. 85(5), 3294–3302 (2003).
[CrossRef] [PubMed]

N. L. Thompson, T. P. Burghardt, and D. Axelrod, “Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy,” Biophys. J. 33(3), 435–454 (1981).
[CrossRef] [PubMed]

Tokunaga, M.

M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, and T. Yanagida, “Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy,” Biochem. Biophys. Res. Commun. 235(1), 47–53 (1997).
[CrossRef] [PubMed]

Truskey, G. A.

A. B. Mathur, G. A. Truskey, and W. M. Reichert, “Atomic force and total internal reflection fluorescence microscopy for the study of force transmission in endothelial cells,” Biophys. J. 78(4), 1725–1735 (2000).
[CrossRef] [PubMed]

G. A. Truskey, J. S. Burmeister, E. Grapa, and W. M. Reichert, “Total internal reflection fluorescence microscopy (TIRFM). II. Topographical mapping of relative cell/substratum separation distances,” J. Cell Sci. 103(Pt 2), 491–499 (1992).
[PubMed]

Ullal, C. K.

B. Harke, C. K. Ullal, J. Keller, and S. W. Hell, “Three-dimensional nanoscopy of colloidal crystals,” Nano Lett. 8(5), 1309–1313 (2008).
[CrossRef] [PubMed]

Verdes, D.

von der Hocht, I.

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[CrossRef] [PubMed]

von Middendorff, C.

C. Ringemann, B. Harke, C. von Middendorff, R. Medda, A. Honigmann, R. Wagner, M. Leutenegger, A. Schönle, S. W. Hell, and C. Eggeling, “Exploring single-molecule dynamics with fluorescence nanoscopy,” New J. Phys. 11(10), 103054 (2009).
[CrossRef]

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Wagner, R.

A. Honigmann, C. Walter, F. Erdmann, C. Eggeling, and R. Wagner, “Characterization of horizontal lipid bilayers as a model system to study lipid phase separation,” Biophys. J. 98(12), 2886–2894 (2010).
[CrossRef] [PubMed]

C. Ringemann, B. Harke, C. von Middendorff, R. Medda, A. Honigmann, R. Wagner, M. Leutenegger, A. Schönle, S. W. Hell, and C. Eggeling, “Exploring single-molecule dynamics with fluorescence nanoscopy,” New J. Phys. 11(10), 103054 (2009).
[CrossRef]

Walter, C.

A. Honigmann, C. Walter, F. Erdmann, C. Eggeling, and R. Wagner, “Characterization of horizontal lipid bilayers as a model system to study lipid phase separation,” Biophys. J. 98(12), 2886–2894 (2010).
[CrossRef] [PubMed]

Webb, W. W.

D. Magde, W. W. Webb, and E. Elson, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[CrossRef]

Weber, W. H.

G. W. Ford and W. H. Weber, “Electromagnetic interactions of molecules with metal surfaces,” Phys. Rep. 113(4), 195–287 (1984).
[CrossRef]

Welford, W. T.

Westphal, V.

E. Rittweger, B. R. Rankin, V. Westphal, and S. W. Hell, “Fluorescence depletion mechanisms in super-resolving STED microscopy,” Chem. Phys. Lett. 442(4-6), 483–487 (2007).
[CrossRef]

V. Westphal and S. W. Hell, “Nanoscale resolution in the focal plane of an optical microscope,” Phys. Rev. Lett. 94(14), 143903 (2005).
[CrossRef] [PubMed]

Wichmann, J.

Widengren, J.

J. Widengren, U. 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]

Wiggins, T. A.

Wildanger, D.

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. 236(1), 35–43 (2009).
[CrossRef] [PubMed]

Willig, K. I.

K. I. Willig, J. Keller, M. Bossi, and S. W. Hell, “STED microscopy resolves nanoparticle assemblies,” New J. Phys. 8(6), 106 (2006).
[CrossRef]

Woturski, W.

U. Golebiewska, M. Nyako, W. Woturski, I. Zaitseva, and S. McLaughlin, “Diffusion coefficient of fluorescent phosphatidylinositol 4,5-bisphosphate in the plasma membrane of cells,” Mol. Biol. Cell 19(4), 1663–1669 (2008).
[CrossRef] [PubMed]

Wurm, C. A.

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[CrossRef] [PubMed]

Yanagida, T.

M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, and T. Yanagida, “Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy,” Biochem. Biophys. Res. Commun. 235(1), 47–53 (1997).
[CrossRef] [PubMed]

Zaitseva, I.

U. Golebiewska, M. Nyako, W. Woturski, I. Zaitseva, and S. McLaughlin, “Diffusion coefficient of fluorescent phosphatidylinositol 4,5-bisphosphate in the plasma membrane of cells,” Mol. Biol. Cell 19(4), 1663–1669 (2008).
[CrossRef] [PubMed]

Archiv für Mikroskopische Anatomie

E. Abbe, “Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung,” Archiv für Mikroskopische Anatomie 9(1), 413–418 (1873).
[CrossRef]

Biochem. Biophys. Res. Commun.

M. Tokunaga, K. Kitamura, K. Saito, A. H. Iwane, and T. Yanagida, “Single molecule imaging of fluorophores and enzymatic reactions achieved by objective-type total internal reflection fluorescence microscopy,” Biochem. Biophys. Res. Commun. 235(1), 47–53 (1997).
[CrossRef] [PubMed]

Biophys. J.

A. B. Mathur, G. A. Truskey, and W. M. Reichert, “Atomic force and total internal reflection fluorescence microscopy for the study of force transmission in endothelial cells,” Biophys. J. 78(4), 1725–1735 (2000).
[CrossRef] [PubMed]

N. L. Thompson, T. P. Burghardt, and D. Axelrod, “Measuring surface dynamics of biomolecules by total internal reflection fluorescence with photobleaching recovery or correlation spectroscopy,” Biophys. J. 33(3), 435–454 (1981).
[CrossRef] [PubMed]

K. Hassler, T. Anhut, R. Rigler, M. Gösch, and T. Lasser, “High count rates with total internal reflection fluorescence correlation spectroscopy,” Biophys. J. 88(1), L01–L03 (2005).
[CrossRef] [PubMed]

A. M. Lieto, R. C. Cush, and N. L. Thompson, “Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy,” Biophys. J. 85(5), 3294–3302 (2003).
[CrossRef] [PubMed]

A. Honigmann, C. Walter, F. Erdmann, C. Eggeling, and R. Wagner, “Characterization of horizontal lipid bilayers as a model system to study lipid phase separation,” Biophys. J. 98(12), 2886–2894 (2010).
[CrossRef] [PubMed]

Chem. Phys. Lett.

E. Rittweger, B. R. Rankin, V. Westphal, and S. W. Hell, “Fluorescence depletion mechanisms in super-resolving STED microscopy,” Chem. Phys. Lett. 442(4-6), 483–487 (2007).
[CrossRef]

Chemistry

K. Kolmakov, V. N. Belov, J. Bierwagen, C. Ringemann, V. Müller, C. Eggeling, and S. W. Hell, “Red-emitting rhodamine dyes for fluorescence microscopy and nanoscopy,” Chemistry 16(1), 158–166 (2010).
[CrossRef] [PubMed]

ChemPhysChem

S. Chiantia, J. Ries, N. Kahya, and P. Schwille, “Combined AFM and two-focus SFCS study of raft-exhibiting model membranes,” ChemPhysChem 7(11), 2409–2418 (2006).
[CrossRef] [PubMed]

T. Dertinger, V. Pacheco, I. von der Hocht, R. Hartmann, I. Gregor, and J. Enderlein, “Two-focus fluorescence correlation spectroscopy: A new tool for accurate and absolute diffusion measurements,” ChemPhysChem 8(3), 433–443 (2007).
[CrossRef] [PubMed]

Curr. Opin. Biotechnol.

H. Schneckenburger, “Total internal reflection fluorescence microscopy: technical innovations and novel applications,” Curr. Opin. Biotechnol. 16(1), 13–18 (2005).
[CrossRef] [PubMed]

J. Cell Biol.

D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol. 89(1), 141–145 (1981).
[CrossRef] [PubMed]

J. Cell Sci.

G. A. Truskey, J. S. Burmeister, E. Grapa, and W. M. Reichert, “Total internal reflection fluorescence microscopy (TIRFM). II. Topographical mapping of relative cell/substratum separation distances,” J. Cell Sci. 103(Pt 2), 491–499 (1992).
[PubMed]

J. Microsc.

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. 236(1), 35–43 (2009).
[CrossRef] [PubMed]

J. Opt. Soc. Am.

J. Opt. Soc. Am. A

J. Phys. Chem.

J. Widengren, U. 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]

Langmuir

S. Chiantia, N. Kahya, and P. Schwille, “Dehydration damage of domain-exhibiting supported bilayers: an AFM study on the protective effects of disaccharides and other stabilizing substances,” Langmuir 21(14), 6317–6323 (2005).
[CrossRef] [PubMed]

Mol. Biol. Cell

U. Golebiewska, M. Nyako, W. Woturski, I. Zaitseva, and S. McLaughlin, “Diffusion coefficient of fluorescent phosphatidylinositol 4,5-bisphosphate in the plasma membrane of cells,” Mol. Biol. Cell 19(4), 1663–1669 (2008).
[CrossRef] [PubMed]

Nano Lett.

B. Harke, C. K. Ullal, J. Keller, and S. W. Hell, “Three-dimensional nanoscopy of colloidal crystals,” Nano Lett. 8(5), 1309–1313 (2008).
[CrossRef] [PubMed]

Nat. Methods

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[CrossRef] [PubMed]

Nature

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

New J. Phys.

C. Ringemann, B. Harke, C. von Middendorff, R. Medda, A. Honigmann, R. Wagner, M. Leutenegger, A. Schönle, S. W. Hell, and C. Eggeling, “Exploring single-molecule dynamics with fluorescence nanoscopy,” New J. Phys. 11(10), 103054 (2009).
[CrossRef]

K. I. Willig, J. Keller, M. Bossi, and S. W. Hell, “STED microscopy resolves nanoparticle assemblies,” New J. Phys. 8(6), 106 (2006).
[CrossRef]

Opt. Express

Opt. Lett.

Phys. Rep.

G. W. Ford and W. H. Weber, “Electromagnetic interactions of molecules with metal surfaces,” Phys. Rep. 113(4), 195–287 (1984).
[CrossRef]

Phys. Rev. A

D. E. Koppel, “Statistical accuracy in fluorescence correlation spectroscopy,” Phys. Rev. A 10(6), 1938–1945 (1974).
[CrossRef]

Phys. Rev. Lett.

R. Dorn, S. Quabis, and G. Leuchs, “Sharper focus for a radially polarized light beam,” Phys. Rev. Lett. 91(23), 233901 (2003).
[CrossRef] [PubMed]

L. Kastrup, H. Blom, C. Eggeling, and S. W. Hell, “Fluorescence fluctuation spectroscopy in subdiffraction focal volumes,” Phys. Rev. Lett. 94(17), 178104 (2005).
[CrossRef] [PubMed]

D. Magde, W. W. Webb, and E. Elson, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
[CrossRef]

V. Westphal and S. W. Hell, “Nanoscale resolution in the focal plane of an optical microscope,” Phys. Rev. Lett. 94(14), 143903 (2005).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. U.S.A.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

Q. Rev. Biophys.

M. Ehrenberg and R. Rigler, “Fluorescence correlation spectroscopy applied to rotational diffusion of macromolecules,” Q. Rev. Biophys. 9(1), 69–81 (1976).
[CrossRef] [PubMed]

Science

S. W. Hell, “Far-field optical nanoscopy,” Science 316(5828), 1153–1158 (2007).
[CrossRef] [PubMed]

Traffic

D. Axelrod, “Total internal reflection fluorescence microscopy in cell biology,” Traffic 2(11), 764–774 (2001).
[CrossRef] [PubMed]

Other

K. Hassler, Single molecule detection and fluorescence correlation spectroscopy on surfaces, doctoral thesis, École Polytechnique Fédérale de Lausanne, Switzerland (2005): http://library.epfl.ch/theses/?nr=3433 .

D. Axelrod, E. H. Hellen, and R. M. Fulbright, “Total internal reflection fluorescence” in Topics in Fluorescence Spectroscopy, J. R. Lakowicz (ed.) (Springer 2002), Vol. 3, pp. 289–343.

J. B. Pawley, Handbook of Biological Confocal Microscopy, 3rd ed. (Springer, New York 2005).

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 (10)

Fig. 1
Fig. 1

Confocal epi-TIRF-STED setup. Lenses are indicated by white ellipsoids. The distance f indicates the focal length of the tube lens between the phase ramp and the tangential polarization controller. Insets show the calculated lateral two-dimensional profiles (2 × 2 μm2) in the plane 5 nm above the cover slip surface of the excitation intensity with tangential polarization, of the STED intensity distribution with circular polarization (left half: laser intensity, right: fluorescence inhibition efficiency at high power), of the detection efficiency and of the brightness of the detected fluorescence (left half: no STED; right: high STED power).

Fig. 2
Fig. 2

Evanescent excitation foci for (a) radially and (b) tangentially polarized light. Calculated cross-sections along the lateral x direction of the evanescent excitation profiles created with an annular illumination of the objective aperture, such that only super-critical angles are illuminated. Shown are the total intensity I (black line) and the relative intensities of the lateral x- (red circles) and y- (green circles) and the axial z-polarization (blue squares) components. The wavelength and power of the laser and the refractive index n of the sample were set to λex = 640 nm, 1 mW and n = 1.33 (water). The insets show the lateral two-dimensional intensity distributions at the cover slip–sample interface (red: x-, green: y- and blue: z-polarized component). (a) Along the x-axis, the radial polarization leads to an overwhelming z-, a zero y- and a small x-polarized intensity component, which has a central intensity minimum. (b) Tangential polarization results in x- and y-polarized intensity components of similar amplitude and a zero z-polarized component.

Fig. 3
Fig. 3

Donut-shaped non-evanescent STED foci for (a) circular and (b) tangentially polarized light. Calculated cross-sections along the lateral x direction of the non-evanescent intensity profiles of the STED foci created by overfilling the objective aperture 1.7 × . Shown are the total intensity I (black line) and the relative intensities of the lateral x- (red circles) and y- (green circles) and the axial z-polarization (blue squares) components. The wavelength and power of the STED laser and the refractive index n of the sample were set to λSTED = 780 nm, 1 mW and n = 1.33 (water). The insets show the intensity distributions at the cover slip–sample interface (red: x-, green: y- and blue: z-polarized component). With tangential polarization, the y-polarized component contributes 100% of the intensity along the x-axis and vice versa, while a z-polarized component is absent.

Fig. 4
Fig. 4

Calculated e−1, e−2 and e−3 iso-surfaces of (a) the normalized evanescent excitation intensity Iex (λex = 640nm, tangential polarization), (b) the normalized donut-shaped STED intensity ISTED (λSTED = 780nm, circular polarization) and of (c) the normalized detection efficiency Q (470nm projected pinhole diameter). The polarization of the laser foci (a,b) is color-coded in red, green and blue representing the x, y and z components, respectively. The refractive index n of the sample was set to 1.33 (water).

Fig. 5
Fig. 5

Calculated normalized brightness profiles of the detected fluorescence emitted by Atto647 fluorophores dissolved in PBS buffer, (a) without STED and (b) with STED, respectively: lateral cross-section along the y-axis and contour lines along the axial z-penetration. The contour lines show the relative brightness in percent of the peak brightness at the focal center. The cover glass surface is at z = 0. (b) The STED beam had a pulse width τSTED = 110 ps at a pulse rate of 79.3 MHz and a power to stimulate the emission 500 × faster than spontaneous decay at the crest of the donut. A STED pulse delay of τΔ = 30 ps and a vibrational relaxation rate kvib = 5/ps of Atto647 were assumed in this calculation.

Fig. 6
Fig. 6

Experimental profiles of (a) the excitation intensity × detection efficiency IexQ, (b) the STED intensity ISTED and (c) their overlap measured with a ∅80 nm gold bead immobilized on the cover slip surface. The PSFs were measured simultaneously by recording (a) the luminescence of the gold bead on the confocal fluorescence detector and (b) the back-scattered light on a non-confocal detector (PMT). (c) The dotted circle illustrates the projected confocal pinhole. The gold bead was mounted in immersion oil to minimize back-reflections of the laser light. Consequently, no evanescent field is created for the excitation due to the mounting in oil, but only the alignment and quality of the excitation PSF are probed. Scale bars: 1 μm.

Fig. 7
Fig. 7

Scanning images (normalized brightness) of ∅20 nm fluorescent beads immobilized on the cover slip for different STED powers of (a) 10 mW, (b) 2 mW, (c) 40 mW and (d) 160 mW following TIRF excitation with tangential polarization and donut shaped STED beam with circular polarization. Area: 10 × 10 μm2, scanning step size 10 nm and dwell time 100 μs. (a), (c), (b) and (d) show the same sample areas.

Fig. 8
Fig. 8

Measured laser intensity profiles (A-C) and resulting TIRF-STED images (normalized brightness) of ∅20 nm fluorescent beads immobilized on the cover slip (a-c). The excitation light was polarized (A,a) radially, (B,b) linearly along the y-axis and (C,c) circularly, respectively. (A-C) The upper and left panels show the experimentally determined xy, xz and yz cross-sections of the excitation × detection profiles (as for Fig. 6 no evanescent field is created due to the mounting in oil), and the lower right panels the lateral xy cross-sections of the STED intensity profile. The circles indicate the size of the confocal pinhole as in Fig. 6. The images (a-c) show the diffraction limited performance in the outer parts and the TIRF-STED performance in the centers (STED power of 160 mW). Scale bars: 1 μm.

Fig. 9
Fig. 9

TIRF-STED-FCS. Normalized measured (colored markers) and fitted (black lines) auto-correlation curves for (a) DOPE–Atto647N diffusing in a DOPC SLB measured with STED powers PSTED in the sample of {0, 11, 18, 26, 35, 53, 70, 105, 140, 210, 280} mW and (b) a 0.5 µM solution of Atto647 in PBS buffer measured with PSTED = {0, 7, 18, 35, 70} mW. The FCS measurements lasted 30s each. The extracted fit parameters are: (a) D ≈5.0 μm2/s, Ps ≈8.8 mW, and FWHM ≈{157, 80, 39} nm at PSTED = {0, 53, 280} mW, respectively, and (b) D ≈156 um2/s, Ps ≈2.7 mW, FWHM ≈{156, 91, 53} nm at PSTED = {0, 18, 70} mW, respectively.

Fig. 10
Fig. 10

STED-FCS in the presence of out-of-focus background. Correlation amplitudes Gd versus transit times τd for various STED powers PSTED for (a) TIRF-STED-FCS and (b) confocal STED-FCS. All parameters were extracted using the fit model (2) for anomalous diffusion in two dimensions. Rectangles indicate the 95% confidence intervals of the fitted parameters (least squares fit). Thin lines show the (partial) linear regressions of Gd(τd). Ideally, a linear correlation between log(Gd) and log(τd) with a slope of –1 is expected. A shallower slope indicates a reduction of the SBR with increasing STED power.

Equations (6)

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

G i (τ)= G i + g(Dτ, P STEDi / P s , τ Δ ) C i ( 1+ p t 1 p t exp( τ τ t ) )
G i (τ)= G i + G di 1+ ( τ/ τ di ) α i ( 1+ p ti 1 p ti exp( τ τ ti ) )
G(τ)= ( Tτ ) 0 Tτ I(t)I(T+τ)dt ( 0 Tτ I(t)dt )( τ T I(t)dt ) =1+ B( r )B( r ')φ( r , r ',τ)d r d r ' ( C B( r )d r ) 2 ,
G(τ)=1+ ( 4πDτ ) d/2 C exp( ρ 2 /( 4Dτ ) ) B( r )B( r + ρ ) d r d ρ ( B( r )d r ) 2 .
g(Dτ)= ( 4πDτ ) d/2 ( B( r )d r ) 2 exp( ρ 2 /( 4Dτ ) )W(ρ)dρ .
G(τ)= G + G d (τ) G t (τ)= G + g(Dτ) C ( 1+ p t 1 p t exp( τ τ t ) ),

Metrics