Abstract

Stimulated emission depletion (STED) resolves fluorescent features that are closer than the far-field optical diffraction limit by applying a spatially modulated light field keeping all but one of these features dark consecutively. For estimating the efficiency of transient fluorophore darkening, we developed analytical equations considering the spatio-temporal intensity profile of the STED beam. These equations provide a quick analysis and optimization of the resolution and contrast to be gained under various conditions, such as continuous wave or pulsed STED beams having different pulse durations. Particular emphasis is placed on fluorescence fluctuation methods such as correlation spectroscopy (FCS) using STED.

© 2010 OSA

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  1. 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]
  2. 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]
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  4. A. Thomas, Progress in Stimulated Emission Depletion Microscopy (Shaker, Achen 2001); Ph.D. thesis, Heidelberg (2001).
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    [CrossRef] [PubMed]
  7. The actual number of de-excitation events is limited by the number of excitations per pulse.
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    [CrossRef]
  9. 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]
  10. V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, “Laser-diode-stimulated emission depletion microscopy,” Appl. Phys. Lett. 82(18), 3125–3127 (2003).
    [CrossRef]
  11. M. Dyba and S. W. Hell, “Photostability of a fluorescent marker under pulsed excited-state depletion through stimulated emission,” Appl. Opt. 42(25), 5123–5129 (2003).
    [CrossRef] [PubMed]
  12. C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: Evidence of two-step photolysis,” Anal. Chem. 70(13), 2651–2659 (1998).
    [CrossRef] [PubMed]
  13. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
    [CrossRef] [PubMed]
  14. K. I. Willig, J. Keller, M. Bossi, and S. W. Hell, “STED microscopy resolves nanoparticle assemblies,” N. J. Phys. 8(6), 106 (2006).
    [CrossRef]
  15. M. Leutenegger, R. Rao, R. A. Leitgeb, and T. Lasser, “Fast focus field calculations,” Opt. Express 14(23), 11277–11291 (2006).
    [CrossRef] [PubMed]
  16. M. Leutenegger and T. Lasser, “Detection efficiency in total internal reflection fluorescence microscopy,” Opt. Express 16(12), 8519–8531 (2008).
    [CrossRef] [PubMed]
  17. K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
    [CrossRef] [PubMed]
  18. S. W. Hell, “Toward fluorescence nanoscopy,” Nat. Biotechnol. 21(11), 1347–1355 (2003).
    [CrossRef] [PubMed]
  19. X. Hao, C. Kuang, T. Wang, and X. Liu, “Effects of polarization on the de-excitation dark focal spot in STED microscopy,” J. Opt. 12(11), 115707 (2010).
    [CrossRef]
  20. D. Magde, E. Elson, and W. Webb, “Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy,” Phys. Rev. Lett. 29(11), 705–708 (1972).
    [CrossRef]
  21. R. Rigler, and E. S. Elson, Fluorescence Correlation Spectroscopy: Theory and Applications, Springer Ser. Chem. Phys. 65, Berlin Heidelberg, Germany (2001).
  22. 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]
  23. D. E. Koppel, “Statistical Accuracy in Fluorescence Correlation Spectroscopy,” Phys. Rev. A 10(6), 1938–1945 (1974).
    [CrossRef]
  24. In case of a long-lived excited state, i.e. ≈12ns in a nano-diamond nitrogen vacancy center [25], the required CW power is only about twice the average power for pulsed operation.
  25. K. Y. Han, K. I. Willig, E. Rittweger, F. Jelezko, C. Eggeling, and S. W. Hell, “Three-dimensional stimulated emission depletion microscopy of nitrogen-vacancy centers in diamond using continuous-wave light,” Nano Lett. 9(9), 3323–3329 (2009).
    [CrossRef] [PubMed]
  26. E. Auksorius, B. R. Boruah, C. Dunsby, P. M. P. Lanigan, G. Kennedy, M. A. A. Neil, and P. M. W. French, “Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging,” Opt. Lett. 33(2), 113–115 (2008).
    [CrossRef] [PubMed]

2010 (1)

X. Hao, C. Kuang, T. Wang, and X. Liu, “Effects of polarization on the de-excitation dark focal spot in STED microscopy,” J. Opt. 12(11), 115707 (2010).
[CrossRef]

2009 (2)

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,” N. J. Phys. 11(10), 103054 (2009).
[CrossRef]

K. Y. Han, K. I. Willig, E. Rittweger, F. Jelezko, C. Eggeling, and S. W. Hell, “Three-dimensional stimulated emission depletion microscopy of nitrogen-vacancy centers in diamond using continuous-wave light,” Nano Lett. 9(9), 3323–3329 (2009).
[CrossRef] [PubMed]

2008 (3)

E. Auksorius, B. R. Boruah, C. Dunsby, P. M. P. Lanigan, G. Kennedy, M. A. A. Neil, and P. M. W. French, “Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging,” Opt. Lett. 33(2), 113–115 (2008).
[CrossRef] [PubMed]

B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16(6), 4154–4162 (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]

2007 (2)

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[CrossRef] [PubMed]

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

2006 (2)

K. I. Willig, J. Keller, M. Bossi, and S. W. Hell, “STED microscopy resolves nanoparticle assemblies,” N. 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]

2005 (2)

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]

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]

2003 (3)

V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, “Laser-diode-stimulated emission depletion microscopy,” Appl. Phys. Lett. 82(18), 3125–3127 (2003).
[CrossRef]

M. Dyba and S. W. Hell, “Photostability of a fluorescent marker under pulsed excited-state depletion through stimulated emission,” Appl. Opt. 42(25), 5123–5129 (2003).
[CrossRef] [PubMed]

S. W. Hell, “Toward fluorescence nanoscopy,” Nat. Biotechnol. 21(11), 1347–1355 (2003).
[CrossRef] [PubMed]

2000 (1)

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]

1998 (1)

C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: Evidence of two-step photolysis,” Anal. Chem. 70(13), 2651–2659 (1998).
[CrossRef] [PubMed]

1994 (1)

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]

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

1974 (1)

D. E. Koppel, “Statistical Accuracy in Fluorescence Correlation Spectroscopy,” Phys. Rev. A 10(6), 1938–1945 (1974).
[CrossRef]

1972 (1)

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

Auksorius, E.

E. Auksorius, B. R. Boruah, C. Dunsby, P. M. P. Lanigan, G. Kennedy, M. A. A. Neil, and P. M. W. French, “Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging,” Opt. Lett. 33(2), 113–115 (2008).
[CrossRef] [PubMed]

Blanca, C. M.

V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, “Laser-diode-stimulated emission depletion microscopy,” Appl. Phys. Lett. 82(18), 3125–3127 (2003).
[CrossRef]

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]

Boruah, B. R.

E. Auksorius, B. R. Boruah, C. Dunsby, P. M. P. Lanigan, G. Kennedy, M. A. A. Neil, and P. M. W. French, “Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging,” Opt. Lett. 33(2), 113–115 (2008).
[CrossRef] [PubMed]

Bossi, M.

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

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Dunsby, C.

E. Auksorius, B. R. Boruah, C. Dunsby, P. M. P. Lanigan, G. Kennedy, M. A. A. Neil, and P. M. W. French, “Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging,” Opt. Lett. 33(2), 113–115 (2008).
[CrossRef] [PubMed]

Dyba, M.

M. Dyba and S. W. Hell, “Photostability of a fluorescent marker under pulsed excited-state depletion through stimulated emission,” Appl. Opt. 42(25), 5123–5129 (2003).
[CrossRef] [PubMed]

V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, “Laser-diode-stimulated emission depletion microscopy,” Appl. Phys. Lett. 82(18), 3125–3127 (2003).
[CrossRef]

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.

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,” N. J. Phys. 11(10), 103054 (2009).
[CrossRef]

K. Y. Han, K. I. Willig, E. Rittweger, F. Jelezko, C. Eggeling, and S. W. Hell, “Three-dimensional stimulated emission depletion microscopy of nitrogen-vacancy centers in diamond using continuous-wave light,” Nano Lett. 9(9), 3323–3329 (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]

C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: Evidence of two-step photolysis,” Anal. Chem. 70(13), 2651–2659 (1998).
[CrossRef] [PubMed]

Egner, 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]

Elson, E.

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

French, P. M. W.

E. Auksorius, B. R. Boruah, C. Dunsby, P. M. P. Lanigan, G. Kennedy, M. A. A. Neil, and P. M. W. French, “Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging,” Opt. Lett. 33(2), 113–115 (2008).
[CrossRef] [PubMed]

Han, K. Y.

K. Y. Han, K. I. Willig, E. Rittweger, F. Jelezko, C. Eggeling, and S. W. Hell, “Three-dimensional stimulated emission depletion microscopy of nitrogen-vacancy centers in diamond using continuous-wave light,” Nano Lett. 9(9), 3323–3329 (2009).
[CrossRef] [PubMed]

Hao, X.

X. Hao, C. Kuang, T. Wang, and X. Liu, “Effects of polarization on the de-excitation dark focal spot in STED microscopy,” J. Opt. 12(11), 115707 (2010).
[CrossRef]

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,” N. J. Phys. 11(10), 103054 (2009).
[CrossRef]

B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16(6), 4154–4162 (2008).
[CrossRef] [PubMed]

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[CrossRef] [PubMed]

Hell, S. W.

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,” N. J. Phys. 11(10), 103054 (2009).
[CrossRef]

K. Y. Han, K. I. Willig, E. Rittweger, F. Jelezko, C. Eggeling, and S. W. Hell, “Three-dimensional stimulated emission depletion microscopy of nitrogen-vacancy centers in diamond using continuous-wave light,” Nano Lett. 9(9), 3323–3329 (2009).
[CrossRef] [PubMed]

B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16(6), 4154–4162 (2008).
[CrossRef] [PubMed]

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

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[CrossRef] [PubMed]

K. I. Willig, J. Keller, M. Bossi, and S. W. Hell, “STED microscopy resolves nanoparticle assemblies,” N. 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]

M. Dyba and S. W. Hell, “Photostability of a fluorescent marker under pulsed excited-state depletion through stimulated emission,” Appl. Opt. 42(25), 5123–5129 (2003).
[CrossRef] [PubMed]

S. W. Hell, “Toward fluorescence nanoscopy,” Nat. Biotechnol. 21(11), 1347–1355 (2003).
[CrossRef] [PubMed]

V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, “Laser-diode-stimulated emission depletion microscopy,” Appl. Phys. Lett. 82(18), 3125–3127 (2003).
[CrossRef]

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]

Honigmann, 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,” N. J. Phys. 11(10), 103054 (2009).
[CrossRef]

Jakobs, S.

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]

Jelezko, F.

K. Y. Han, K. I. Willig, E. Rittweger, F. Jelezko, C. Eggeling, and S. W. Hell, “Three-dimensional stimulated emission depletion microscopy of nitrogen-vacancy centers in diamond using continuous-wave light,” Nano Lett. 9(9), 3323–3329 (2009).
[CrossRef] [PubMed]

Kastrup, L.

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]

V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, “Laser-diode-stimulated emission depletion microscopy,” Appl. Phys. Lett. 82(18), 3125–3127 (2003).
[CrossRef]

Keller, J.

B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16(6), 4154–4162 (2008).
[CrossRef] [PubMed]

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

Kennedy, G.

E. Auksorius, B. R. Boruah, C. Dunsby, P. M. P. Lanigan, G. Kennedy, M. A. A. Neil, and P. M. W. French, “Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging,” Opt. Lett. 33(2), 113–115 (2008).
[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]

Koppel, D. E.

D. E. Koppel, “Statistical Accuracy in Fluorescence Correlation Spectroscopy,” Phys. Rev. A 10(6), 1938–1945 (1974).
[CrossRef]

Kuang, C.

X. Hao, C. Kuang, T. Wang, and X. Liu, “Effects of polarization on the de-excitation dark focal spot in STED microscopy,” J. Opt. 12(11), 115707 (2010).
[CrossRef]

Lanigan, P. M. P.

E. Auksorius, B. R. Boruah, C. Dunsby, P. M. P. Lanigan, G. Kennedy, M. A. A. Neil, and P. M. W. French, “Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging,” Opt. Lett. 33(2), 113–115 (2008).
[CrossRef] [PubMed]

Lasser, T.

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

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

Leitgeb, R. A.

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

Leutenegger, M.

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,” N. J. Phys. 11(10), 103054 (2009).
[CrossRef]

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

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

Liu, X.

X. Hao, C. Kuang, T. Wang, and X. Liu, “Effects of polarization on the de-excitation dark focal spot in STED microscopy,” J. Opt. 12(11), 115707 (2010).
[CrossRef]

Magde, D.

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

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,” N. J. Phys. 11(10), 103054 (2009).
[CrossRef]

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[CrossRef] [PubMed]

Neil, M. A. A.

E. Auksorius, B. R. Boruah, C. Dunsby, P. M. P. Lanigan, G. Kennedy, M. A. A. Neil, and P. M. W. French, “Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging,” Opt. Lett. 33(2), 113–115 (2008).
[CrossRef] [PubMed]

Rao, R.

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

Rigler, R.

C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: Evidence of two-step photolysis,” Anal. Chem. 70(13), 2651–2659 (1998).
[CrossRef] [PubMed]

Ringemann, 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,” N. J. Phys. 11(10), 103054 (2009).
[CrossRef]

Rittweger, E.

K. Y. Han, K. I. Willig, E. Rittweger, F. Jelezko, C. Eggeling, and S. W. Hell, “Three-dimensional stimulated emission depletion microscopy of nitrogen-vacancy centers in diamond using continuous-wave light,” Nano Lett. 9(9), 3323–3329 (2009).
[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,” N. J. Phys. 11(10), 103054 (2009).
[CrossRef]

B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16(6), 4154–4162 (2008).
[CrossRef] [PubMed]

Seidel, C. A. M.

C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: Evidence of two-step photolysis,” Anal. Chem. 70(13), 2651–2659 (1998).
[CrossRef] [PubMed]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Ullal, C. K.

B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16(6), 4154–4162 (2008).
[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,” N. J. Phys. 11(10), 103054 (2009).
[CrossRef]

Wagner, 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,” N. J. Phys. 11(10), 103054 (2009).
[CrossRef]

Wang, T.

X. Hao, C. Kuang, T. Wang, and X. Liu, “Effects of polarization on the de-excitation dark focal spot in STED microscopy,” J. Opt. 12(11), 115707 (2010).
[CrossRef]

Webb, W.

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

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Westphal, V.

B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16(6), 4154–4162 (2008).
[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]

V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, “Laser-diode-stimulated emission depletion microscopy,” Appl. Phys. Lett. 82(18), 3125–3127 (2003).
[CrossRef]

Wichmann, J.

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]

Widengren, J.

C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: Evidence of two-step photolysis,” Anal. Chem. 70(13), 2651–2659 (1998).
[CrossRef] [PubMed]

Willig, K. I.

K. Y. Han, K. I. Willig, E. Rittweger, F. Jelezko, C. Eggeling, and S. W. Hell, “Three-dimensional stimulated emission depletion microscopy of nitrogen-vacancy centers in diamond using continuous-wave light,” Nano Lett. 9(9), 3323–3329 (2009).
[CrossRef] [PubMed]

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[CrossRef] [PubMed]

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

Anal. Chem. (1)

C. Eggeling, J. Widengren, R. Rigler, and C. A. M. Seidel, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: Evidence of two-step photolysis,” Anal. Chem. 70(13), 2651–2659 (1998).
[CrossRef] [PubMed]

Appl. Opt. (1)

M. Dyba and S. W. Hell, “Photostability of a fluorescent marker under pulsed excited-state depletion through stimulated emission,” Appl. Opt. 42(25), 5123–5129 (2003).
[CrossRef] [PubMed]

Appl. Phys. Lett. (1)

V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, “Laser-diode-stimulated emission depletion microscopy,” Appl. Phys. Lett. 82(18), 3125–3127 (2003).
[CrossRef]

J. Opt. (1)

X. Hao, C. Kuang, T. Wang, and X. Liu, “Effects of polarization on the de-excitation dark focal spot in STED microscopy,” J. Opt. 12(11), 115707 (2010).
[CrossRef]

N. J. Phys. (2)

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

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,” N. J. Phys. 11(10), 103054 (2009).
[CrossRef]

Nano Lett. (1)

K. Y. Han, K. I. Willig, E. Rittweger, F. Jelezko, C. Eggeling, and S. W. Hell, “Three-dimensional stimulated emission depletion microscopy of nitrogen-vacancy centers in diamond using continuous-wave light,” Nano Lett. 9(9), 3323–3329 (2009).
[CrossRef] [PubMed]

Nat. Biotechnol. (1)

S. W. Hell, “Toward fluorescence nanoscopy,” Nat. Biotechnol. 21(11), 1347–1355 (2003).
[CrossRef] [PubMed]

Nat. Methods (1)

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[CrossRef] [PubMed]

Opt. Express (3)

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

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

B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16(6), 4154–4162 (2008).
[CrossRef] [PubMed]

Opt. Lett. (2)

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]

E. Auksorius, B. R. Boruah, C. Dunsby, P. M. P. Lanigan, G. Kennedy, M. A. A. Neil, and P. M. W. French, “Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging,” Opt. Lett. 33(2), 113–115 (2008).
[CrossRef] [PubMed]

Phys. Rev. A (1)

D. E. Koppel, “Statistical Accuracy in Fluorescence Correlation Spectroscopy,” Phys. Rev. A 10(6), 1938–1945 (1974).
[CrossRef]

Phys. Rev. Lett. (3)

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

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]

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. (1)

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]

Science (2)

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

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Other (5)

A. Thomas, Progress in Stimulated Emission Depletion Microscopy (Shaker, Achen 2001); Ph.D. thesis, Heidelberg (2001).

S. W. Hell, “Increasing the Resolution of Far-Field Fluorescence Microscopy by Point-Spread-Function Engineering,” in Topics In Fluorescence Spectroscopy 5: Nonlinear and Two-Photon-Induced Fluorescence, J. Lakowicz, ed. (Plenum Press, New York, 1997).

The actual number of de-excitation events is limited by the number of excitations per pulse.

R. Rigler, and E. S. Elson, Fluorescence Correlation Spectroscopy: Theory and Applications, Springer Ser. Chem. Phys. 65, Berlin Heidelberg, Germany (2001).

In case of a long-lived excited state, i.e. ≈12ns in a nano-diamond nitrogen vacancy center [25], the required CW power is only about twice the average power for pulsed operation.

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Figures (7)

Fig. 1
Fig. 1

Simplified Jablonski diagram with electronic and vibrational energy levels of a typical fluorophore.

Fig. 2
Fig. 2

Probability of spontaneous decay η ps as a function of the saturation factor ζ and the pulse width τ STED of the STED light (as labeled) for k S1 = 1/3.9ns, k vib = 5/ps and T = 1/80MHz. (a) The thin line shows the lower limit achieved for CW STED and infinitely fast vibrational relaxation. Thin dashed lines show the dependence of η ps on variable pulse lengths τ STED for fixed numbers of potential de-excitations k STED τ STED, i.e. for fixed average STED powers. (b) This dependence is shown as a function of τ STED. The inset outlines the pulse scheme used in the calculations: a Dirac excitation pulse followed by a rectangular STED pulse.

Fig. 3
Fig. 3

Cross-sections through the foci of the excitation and the STED light spots (a) and of the fluorescence collection efficiency (b) used in the calculation examples. Insets show the spot profiles in the xy-planes.

Fig. 4
Fig. 4

Effective PSF of a STED nanoscope with (a) pulsed (τ STED = 50ps) and (b) CW STED (τ STED = T = 12.5ns): lateral cross-sections of the detected fluorescence brightness without STED (bold solid lines) and with average STED powers required for achieving FWHM diameters of 72nm (thin solid lines) and 33nm (pointed red lines). Dash-dotted lines show the profiles with an imperfect “zero” and dotted lines show them normalized again to their peak brightness. For values below 10% of the peak brightness, the logarithmic scale allows a better distinction of the differences.

Fig. 5
Fig. 5

FWHM diameters versus STED power. For k STED τ STED > 500 in case of 50ps STED pulses with ε = 1%, the increase of the FWHM indicates loss of signal due to a too strong reduction of the central peak brightness versus the contributions from the focal periphery.

Fig. 6
Fig. 6

(a) SBR at 10% of maximum threshold. (b) RHS diameters (thin lines: FWHM).

Fig. 7
Fig. 7

Probability of spontaneous decay η ps obtained with Eq. (3) compared to the exact results based on Eq. (13) for the very same parameters as described in Fig. 2. The exact results are overlaid as thin lines and are in excellent agreement except at STED pulse widths τ STED < 10ps.

Tables (1)

Tables Icon

Table 1 STED efficiency η ps = n S1(γ)/n S1(0) with instantaneous excitation and pulsed or continuous STED irradiation.

Equations (15)

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t P S1 = k S1 P S1 k STED ( P S1 P S 0 * ) t P S0 * = k vib P S0 * + k STED ( P S1 P S0 * )
F ( γ ) = q fl k S1 0 T P S1 ( t ) d t = q fl ( 1 + γ exp ( k S1 τ STED ( 1 + γ ) ) 1 + γ exp ( k S1 ( γ τ STED + T ) ) ) .
η ps = ( 1 + γ exp ( k S1 τ STED ( 1 + γ ) ) 1 + γ exp ( k S1 ( γ τ STED + T ) ) ) ( 1 exp ( k S1 T ) ) 1
η ps 1 + γ exp ( k S1 τ STED ( 1 + γ ) ) 1 + γ .
η ps 1 / ( 1 + γ ) .
η CW k ex + k S1 k ex + ( 1 + γ ) k S1 .
η ps * = exp ( k S1 τ STED γ ) = exp ( ln ( 2 ) I STED / I s * )
P S0 = 1 P S1 P T1 P S1 + = P S1 + n S0 P S0 P S1 = exp ( k S1 ( γ τ STED + T ) ) P S1 +
P T1 + P T1 + q isc n S1 P S1 + P T1 exp ( k T1 T ) P T1 +
P S1 + = n S0 + ( 1 n S0 ) exp ( k S1 ( γ τ STED + T ) ) P S1 + q isc n S1 n S0 P S1 + exp ( k T1 T ) 1
k ps = n S1 n S0 T ( 1 ( 1 n S0 ) exp ( k S1 ( γ τ STED + T ) ) + q isc n S1 n S0 exp ( k T1 T ) 1 ) 1
n S0 * = k ex * k ex * + k S1 ( 1 + γ ) ( 1 exp ( ( k ex * + k S1 ( 1 + γ ) ) τ ex ) ) .
P S1 ( 0 < t < τ STED ) = k S1 k vib + k 1 2 k 1 exp ( k 2 + k 1 2 t ) k S1 k vib k 1 2 k 1 exp ( k 2 k 1 2 t ) ,
τ CW = 1 k ex * + 1 k S1 + k STED * + q isc * k T1 = 1 k ex * + 1 k S1 ( 1 + γ ) + q isc k T1 ( 1 + γ )
k CW = ( 1 + γ k ex * + 1 k S1 + q isc k T1 ) 1 and η CW = ( 1 k ex * + 1 k S1 + q isc k T1 ) k CW .

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