OSA's Digital Library

Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 20, Iss. 5 — Feb. 27, 2012
  • pp: 5243–5263

Fluorescence correlation spectroscopy with a total internal reflection fluorescence STED microscope (TIRF-STED-FCS)

Marcel Leutenegger, Christian Ringemann, Theo Lasser, Stefan W. Hell, and Christian Eggeling  »View Author Affiliations

Optics Express, Vol. 20, Issue 5, pp. 5243-5263 (2012)

View Full Text Article

Enhanced HTML    Acrobat PDF (5272 KB)

Browse Journals / Lookup Meetings

Browse by Journal and Year


Lookup Conference Papers

Close Browse Journals / Lookup Meetings

Article Tools



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

OCIS Codes
(050.1940) Diffraction and gratings : Diffraction
(180.2520) Microscopy : Fluorescence microscopy
(260.2510) Physical optics : Fluorescence

ToC Category:

Original Manuscript: November 16, 2011
Revised Manuscript: February 9, 2012
Manuscript Accepted: February 10, 2012
Published: February 17, 2012

Virtual Issues
Vol. 7, Iss. 4 Virtual Journal for Biomedical Optics

Marcel Leutenegger, Christian Ringemann, Theo Lasser, Stefan W. Hell, and Christian Eggeling, "Fluorescence correlation spectroscopy with a total internal reflection fluorescence STED microscope (TIRF-STED-FCS)," Opt. Express 20, 5243-5263 (2012)

Sort:  Author  |  Year  |  Journal  |  Reset  


  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 Anatomie9(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,” Science316(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. Methods5(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,” Nature457(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,” Traffic2(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. Express12(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. A8(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. Express19(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. Express14(23), 11277–11291 (2006). [CrossRef] [PubMed]
  33. M. Leutenegger and T. Lasser, “Detection efficiency in total internal reflection fluorescence microscopy,” Opt. Express16(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. Express18(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,” Langmuir21(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,” ChemPhysChem7(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. Cell19(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,” ChemPhysChem8(3), 433–443 (2007). [CrossRef] [PubMed]
  44. D. E. Koppel, “Statistical accuracy in fluorescence correlation spectroscopy,” Phys. Rev. A10(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,” Chemistry16(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. A9(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. A11(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 .

Cited By

Alert me when this paper is cited

OSA is able to provide readers links to articles that cite this paper by participating in CrossRef's Cited-By Linking service. CrossRef includes content from more than 3000 publishers and societies. In addition to listing OSA journal articles that cite this paper, citing articles from other participating publishers will also be listed.

« Previous Article  |  Next Article »

OSA is a member of CrossRef.

CrossCheck Deposited