Abstract

We report on a straightforward yet powerful implementation of stimulated emission depletion (STED) fluorescence microscopy providing subdiffraction resolution in the far-field. Utilizing the same supercontinuum pulsed laser source both for excitation and STED, this implementation of STED microscopy avoids elaborate preparations of laser pulses and conveniently provides multicolor imaging. Operating at pulse repetition rates around 1 MHz, it also affords reduced photobleaching rates by allowing the fluorophore to relax from excitable metastable dark states involved in photodegradation. The imaging of dense nanoparticles and of the microtubular network of mammalian cells evidences a spatial resolution of 30–50 nm in the focal plane, i.e. by a factor of 8–9 beyond the diffraction barrier.

© 2008 Optical Society of America

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  1. S. W. Hell and J. Wichmann, "Breaking the diffraction resolution limit by stimulated emission: stimulated emission depletion microscopy," Opt. Lett. 19, 780-782 (1994).
    [CrossRef] [PubMed]
  2. T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, "Fluorescence microscopy with diffraction resolution limit broken by stimulated emission," Proc. Nat. Acad. Sci. U.S.A. 97, 8206-8210 (2000).
    [CrossRef]
  3. K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, "STED-microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis," Nature 440, 935 - 939 (2006).
    [CrossRef] [PubMed]
  4. G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell "Macromolecular-scale resolution in biological fluorescence microscopy," Proc. Natl. Acad. Sci. U. S. A. 103, 11440-11445 (2006).
    [CrossRef] [PubMed]
  5. Q1. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, "Imaging Intracellular Fluorescent Proteins at Nanometer Resolution," Science 313, 1642-1645 (2006).
    [CrossRef] [PubMed]
  6. M. J. Rust, M. Bates, and X. Zhuang, "Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)," Nat Meth 3, 793-796 (2006).
    [CrossRef]
  7. S. T. Hess, T. P. K. Girirajan, and M. D. Mason, "Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy," Biophys. J. 91, 4258-4272 (2006).
    [CrossRef] [PubMed]
  8. S. W. Hell, "Far-Field Optical Nanoscopy," Science 316, 1153-1158 (2007).
    [CrossRef] [PubMed]
  9. K. I. Willig, B. Harke, R. Medda, and S. W. Hell, "STED microscopy with continuous wave beams," Nature Methods 4, 915-918 (2007).
    [CrossRef] [PubMed]
  10. V. Westphal and S. W. Hell, "Nanoscale Resolution in the Focal Plane of an Optical Microscope," Phys. Rev. Lett. 94, 143903 (2005).
    [CrossRef] [PubMed]
  11. B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schoenle, and S. W. Hell, "Resolution scaling in STED microscopy," Opt. Express 16, 4154-4162 (2008).
    [CrossRef] [PubMed]
  12. E. K. L. Yeow, S. M. Melnikov, T. D. M. Bell, F. C. DeSchryver, and J. Hofkens, "Characterizing the Fluorescence Intermittency and Photobleaching Kinetics of Dye Molecules Immobilized on a Glass Surface," J. Phys. Chem. A 110, 1726-1734 (2006).
    [CrossRef] [PubMed]
  13. R. Zondervan, F. Kulzer, M. A. Kolchenko, and M. Orrit, "Photobleaching of Rhodamine 6G in Poly(vinyl alcohol) at the ensemble and single-molecule levels," J. Phys. Chem. A 108, 1657-1665 (2004).
    [CrossRef]
  14. L. A. Deschenes and D. A. Vanden Bout, "Single molecule photobleaching: increasing photon yield and survival time through suppression of two-step photolysis," Chem. Phys. Lett. 365, 387-395 (2002).
    [CrossRef]
  15. P. Russell, "Photonic Crystal Fibers," Science 299, 358-362 (2003).
    [CrossRef] [PubMed]
  16. J. K. Ranka, R. S. Windeler, and A. J. Stentz, "Visible continuum generation in air-silica microstructure optical fibers with anomalous dispersion at 800 nm," Opt. Lett. 25, 25-27 (2000).
    [CrossRef]
  17. 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, 113-115 (2008).
    [CrossRef] [PubMed]
  18. J. Keller, A. Schönle, and S. W. Hell, "Efficient fluorescence inhibition patterns for RESOLFT microscopy," Opt. Express 15, 3361-3371 (2007).
    [CrossRef] [PubMed]
  19. M. Osborn and K. Weber, "Immunofluorescence and Immunocytochemical Procedures with Affinity Purified Antibodies: Tubulin-Containing Structures," Meth. Cell. Biol. 24, 97-132 (1982).
    [CrossRef]
  20. J. Swartzlander and A. Grover, "Achromatic optical vortex lens," Opt. Lett. 31, 2042-2044 (2006).
    [CrossRef] [PubMed]

2008 (2)

2007 (3)

J. Keller, A. Schönle, and S. W. Hell, "Efficient fluorescence inhibition patterns for RESOLFT microscopy," Opt. Express 15, 3361-3371 (2007).
[CrossRef] [PubMed]

S. W. Hell, "Far-Field Optical Nanoscopy," Science 316, 1153-1158 (2007).
[CrossRef] [PubMed]

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, "STED microscopy with continuous wave beams," Nature Methods 4, 915-918 (2007).
[CrossRef] [PubMed]

2006 (7)

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, "STED-microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis," Nature 440, 935 - 939 (2006).
[CrossRef] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell "Macromolecular-scale resolution in biological fluorescence microscopy," Proc. Natl. Acad. Sci. U. S. A. 103, 11440-11445 (2006).
[CrossRef] [PubMed]

Q1. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, "Imaging Intracellular Fluorescent Proteins at Nanometer Resolution," Science 313, 1642-1645 (2006).
[CrossRef] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, "Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)," Nat Meth 3, 793-796 (2006).
[CrossRef]

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, "Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy," Biophys. J. 91, 4258-4272 (2006).
[CrossRef] [PubMed]

E. K. L. Yeow, S. M. Melnikov, T. D. M. Bell, F. C. DeSchryver, and J. Hofkens, "Characterizing the Fluorescence Intermittency and Photobleaching Kinetics of Dye Molecules Immobilized on a Glass Surface," J. Phys. Chem. A 110, 1726-1734 (2006).
[CrossRef] [PubMed]

J. Swartzlander and A. Grover, "Achromatic optical vortex lens," Opt. Lett. 31, 2042-2044 (2006).
[CrossRef] [PubMed]

2005 (1)

V. Westphal and S. W. Hell, "Nanoscale Resolution in the Focal Plane of an Optical Microscope," Phys. Rev. Lett. 94, 143903 (2005).
[CrossRef] [PubMed]

2004 (1)

R. Zondervan, F. Kulzer, M. A. Kolchenko, and M. Orrit, "Photobleaching of Rhodamine 6G in Poly(vinyl alcohol) at the ensemble and single-molecule levels," J. Phys. Chem. A 108, 1657-1665 (2004).
[CrossRef]

2003 (1)

P. Russell, "Photonic Crystal Fibers," Science 299, 358-362 (2003).
[CrossRef] [PubMed]

2002 (1)

L. A. Deschenes and D. A. Vanden Bout, "Single molecule photobleaching: increasing photon yield and survival time through suppression of two-step photolysis," Chem. Phys. Lett. 365, 387-395 (2002).
[CrossRef]

2000 (2)

J. K. Ranka, R. S. Windeler, and A. J. Stentz, "Visible continuum generation in air-silica microstructure optical fibers with anomalous dispersion at 800 nm," Opt. Lett. 25, 25-27 (2000).
[CrossRef]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, "Fluorescence microscopy with diffraction resolution limit broken by stimulated emission," Proc. Nat. Acad. Sci. U.S.A. 97, 8206-8210 (2000).
[CrossRef]

1994 (1)

1982 (1)

M. Osborn and K. Weber, "Immunofluorescence and Immunocytochemical Procedures with Affinity Purified Antibodies: Tubulin-Containing Structures," Meth. Cell. Biol. 24, 97-132 (1982).
[CrossRef]

Andrei, M. A.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell "Macromolecular-scale resolution in biological fluorescence microscopy," Proc. Natl. Acad. Sci. U. S. A. 103, 11440-11445 (2006).
[CrossRef] [PubMed]

Auksorius, E.

Bates, M.

M. J. Rust, M. Bates, and X. Zhuang, "Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)," Nat Meth 3, 793-796 (2006).
[CrossRef]

Bell, T. D. M.

E. K. L. Yeow, S. M. Melnikov, T. D. M. Bell, F. C. DeSchryver, and J. Hofkens, "Characterizing the Fluorescence Intermittency and Photobleaching Kinetics of Dye Molecules Immobilized on a Glass Surface," J. Phys. Chem. A 110, 1726-1734 (2006).
[CrossRef] [PubMed]

Betzig, E.

Q1. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, "Imaging Intracellular Fluorescent Proteins at Nanometer Resolution," Science 313, 1642-1645 (2006).
[CrossRef] [PubMed]

Bonifacino, J. S.

Q1. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, "Imaging Intracellular Fluorescent Proteins at Nanometer Resolution," Science 313, 1642-1645 (2006).
[CrossRef] [PubMed]

Boruah, B. R.

Davidson, M. W.

Q1. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, "Imaging Intracellular Fluorescent Proteins at Nanometer Resolution," Science 313, 1642-1645 (2006).
[CrossRef] [PubMed]

Deschenes, L. A.

L. A. Deschenes and D. A. Vanden Bout, "Single molecule photobleaching: increasing photon yield and survival time through suppression of two-step photolysis," Chem. Phys. Lett. 365, 387-395 (2002).
[CrossRef]

DeSchryver, F. C.

E. K. L. Yeow, S. M. Melnikov, T. D. M. Bell, F. C. DeSchryver, and J. Hofkens, "Characterizing the Fluorescence Intermittency and Photobleaching Kinetics of Dye Molecules Immobilized on a Glass Surface," J. Phys. Chem. A 110, 1726-1734 (2006).
[CrossRef] [PubMed]

Donnert, G.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell "Macromolecular-scale resolution in biological fluorescence microscopy," Proc. Natl. Acad. Sci. U. S. A. 103, 11440-11445 (2006).
[CrossRef] [PubMed]

Dunsby, C.

Dyba, M.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, "Fluorescence microscopy with diffraction resolution limit broken by stimulated emission," Proc. Nat. Acad. Sci. U.S.A. 97, 8206-8210 (2000).
[CrossRef]

Eggeling, C.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell "Macromolecular-scale resolution in biological fluorescence microscopy," Proc. Natl. Acad. Sci. U. S. A. 103, 11440-11445 (2006).
[CrossRef] [PubMed]

Egner, A.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, "Fluorescence microscopy with diffraction resolution limit broken by stimulated emission," Proc. Nat. Acad. Sci. U.S.A. 97, 8206-8210 (2000).
[CrossRef]

French, P. M. W.

Girirajan, T. P. K.

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, "Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy," Biophys. J. 91, 4258-4272 (2006).
[CrossRef] [PubMed]

Grover, A.

Harke, B.

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

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, "STED microscopy with continuous wave beams," Nature Methods 4, 915-918 (2007).
[CrossRef] [PubMed]

Hell, S. W.

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

S. W. Hell, "Far-Field Optical Nanoscopy," Science 316, 1153-1158 (2007).
[CrossRef] [PubMed]

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, "STED microscopy with continuous wave beams," Nature Methods 4, 915-918 (2007).
[CrossRef] [PubMed]

J. Keller, A. Schönle, and S. W. Hell, "Efficient fluorescence inhibition patterns for RESOLFT microscopy," Opt. Express 15, 3361-3371 (2007).
[CrossRef] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell "Macromolecular-scale resolution in biological fluorescence microscopy," Proc. Natl. Acad. Sci. U. S. A. 103, 11440-11445 (2006).
[CrossRef] [PubMed]

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, "STED-microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis," Nature 440, 935 - 939 (2006).
[CrossRef] [PubMed]

V. Westphal and S. W. Hell, "Nanoscale Resolution in the Focal Plane of an Optical Microscope," Phys. Rev. Lett. 94, 143903 (2005).
[CrossRef] [PubMed]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, "Fluorescence microscopy with diffraction resolution limit broken by stimulated emission," Proc. Nat. Acad. Sci. U.S.A. 97, 8206-8210 (2000).
[CrossRef]

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

Hess, H. F.

Q1. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, "Imaging Intracellular Fluorescent Proteins at Nanometer Resolution," Science 313, 1642-1645 (2006).
[CrossRef] [PubMed]

Hess, S. T.

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, "Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy," Biophys. J. 91, 4258-4272 (2006).
[CrossRef] [PubMed]

Hofkens, J.

E. K. L. Yeow, S. M. Melnikov, T. D. M. Bell, F. C. DeSchryver, and J. Hofkens, "Characterizing the Fluorescence Intermittency and Photobleaching Kinetics of Dye Molecules Immobilized on a Glass Surface," J. Phys. Chem. A 110, 1726-1734 (2006).
[CrossRef] [PubMed]

Jahn, R.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell "Macromolecular-scale resolution in biological fluorescence microscopy," Proc. Natl. Acad. Sci. U. S. A. 103, 11440-11445 (2006).
[CrossRef] [PubMed]

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, "STED-microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis," Nature 440, 935 - 939 (2006).
[CrossRef] [PubMed]

Jakobs, S.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, "Fluorescence microscopy with diffraction resolution limit broken by stimulated emission," Proc. Nat. Acad. Sci. U.S.A. 97, 8206-8210 (2000).
[CrossRef]

Keller, J.

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

J. Keller, A. Schönle, and S. W. Hell, "Efficient fluorescence inhibition patterns for RESOLFT microscopy," Opt. Express 15, 3361-3371 (2007).
[CrossRef] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell "Macromolecular-scale resolution in biological fluorescence microscopy," Proc. Natl. Acad. Sci. U. S. A. 103, 11440-11445 (2006).
[CrossRef] [PubMed]

Kennedy, G.

Klar, T. A.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, "Fluorescence microscopy with diffraction resolution limit broken by stimulated emission," Proc. Nat. Acad. Sci. U.S.A. 97, 8206-8210 (2000).
[CrossRef]

Kolchenko, M. A.

R. Zondervan, F. Kulzer, M. A. Kolchenko, and M. Orrit, "Photobleaching of Rhodamine 6G in Poly(vinyl alcohol) at the ensemble and single-molecule levels," J. Phys. Chem. A 108, 1657-1665 (2004).
[CrossRef]

Kulzer, F.

R. Zondervan, F. Kulzer, M. A. Kolchenko, and M. Orrit, "Photobleaching of Rhodamine 6G in Poly(vinyl alcohol) at the ensemble and single-molecule levels," J. Phys. Chem. A 108, 1657-1665 (2004).
[CrossRef]

Lanigan, P. M. P.

Lindwasser, O. W.

Q1. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, "Imaging Intracellular Fluorescent Proteins at Nanometer Resolution," Science 313, 1642-1645 (2006).
[CrossRef] [PubMed]

Lippincott-Schwartz, J.

Q1. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, "Imaging Intracellular Fluorescent Proteins at Nanometer Resolution," Science 313, 1642-1645 (2006).
[CrossRef] [PubMed]

Lührmann, R.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell "Macromolecular-scale resolution in biological fluorescence microscopy," Proc. Natl. Acad. Sci. U. S. A. 103, 11440-11445 (2006).
[CrossRef] [PubMed]

Mason, M. D.

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, "Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy," Biophys. J. 91, 4258-4272 (2006).
[CrossRef] [PubMed]

Medda, R.

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, "STED microscopy with continuous wave beams," Nature Methods 4, 915-918 (2007).
[CrossRef] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell "Macromolecular-scale resolution in biological fluorescence microscopy," Proc. Natl. Acad. Sci. U. S. A. 103, 11440-11445 (2006).
[CrossRef] [PubMed]

Melnikov, S. M.

E. K. L. Yeow, S. M. Melnikov, T. D. M. Bell, F. C. DeSchryver, and J. Hofkens, "Characterizing the Fluorescence Intermittency and Photobleaching Kinetics of Dye Molecules Immobilized on a Glass Surface," J. Phys. Chem. A 110, 1726-1734 (2006).
[CrossRef] [PubMed]

Neil, M. A. A.

Olenych, S.

Q1. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, "Imaging Intracellular Fluorescent Proteins at Nanometer Resolution," Science 313, 1642-1645 (2006).
[CrossRef] [PubMed]

Orrit, M.

R. Zondervan, F. Kulzer, M. A. Kolchenko, and M. Orrit, "Photobleaching of Rhodamine 6G in Poly(vinyl alcohol) at the ensemble and single-molecule levels," J. Phys. Chem. A 108, 1657-1665 (2004).
[CrossRef]

Osborn, M.

M. Osborn and K. Weber, "Immunofluorescence and Immunocytochemical Procedures with Affinity Purified Antibodies: Tubulin-Containing Structures," Meth. Cell. Biol. 24, 97-132 (1982).
[CrossRef]

Patterson, G. H.

Q1. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, "Imaging Intracellular Fluorescent Proteins at Nanometer Resolution," Science 313, 1642-1645 (2006).
[CrossRef] [PubMed]

Ranka, J. K.

Rizzoli, S. O.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell "Macromolecular-scale resolution in biological fluorescence microscopy," Proc. Natl. Acad. Sci. U. S. A. 103, 11440-11445 (2006).
[CrossRef] [PubMed]

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, "STED-microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis," Nature 440, 935 - 939 (2006).
[CrossRef] [PubMed]

Russell, P.

P. Russell, "Photonic Crystal Fibers," Science 299, 358-362 (2003).
[CrossRef] [PubMed]

Rust, M. J.

M. J. Rust, M. Bates, and X. Zhuang, "Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)," Nat Meth 3, 793-796 (2006).
[CrossRef]

Schoenle, A.

Schönle, A.

Sougrat, R.

Q1. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, "Imaging Intracellular Fluorescent Proteins at Nanometer Resolution," Science 313, 1642-1645 (2006).
[CrossRef] [PubMed]

Stentz, A. J.

Swartzlander, J.

Ullal, C. K.

Vanden Bout, D. A.

L. A. Deschenes and D. A. Vanden Bout, "Single molecule photobleaching: increasing photon yield and survival time through suppression of two-step photolysis," Chem. Phys. Lett. 365, 387-395 (2002).
[CrossRef]

Weber, K.

M. Osborn and K. Weber, "Immunofluorescence and Immunocytochemical Procedures with Affinity Purified Antibodies: Tubulin-Containing Structures," Meth. Cell. Biol. 24, 97-132 (1982).
[CrossRef]

Westphal, V.

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

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, "STED-microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis," Nature 440, 935 - 939 (2006).
[CrossRef] [PubMed]

V. Westphal and S. W. Hell, "Nanoscale Resolution in the Focal Plane of an Optical Microscope," Phys. Rev. Lett. 94, 143903 (2005).
[CrossRef] [PubMed]

Wichmann, J.

Willig, K. I.

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, "STED microscopy with continuous wave beams," Nature Methods 4, 915-918 (2007).
[CrossRef] [PubMed]

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, "STED-microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis," Nature 440, 935 - 939 (2006).
[CrossRef] [PubMed]

Windeler, R. S.

Yeow, E. K. L.

E. K. L. Yeow, S. M. Melnikov, T. D. M. Bell, F. C. DeSchryver, and J. Hofkens, "Characterizing the Fluorescence Intermittency and Photobleaching Kinetics of Dye Molecules Immobilized on a Glass Surface," J. Phys. Chem. A 110, 1726-1734 (2006).
[CrossRef] [PubMed]

Zhuang, X.

M. J. Rust, M. Bates, and X. Zhuang, "Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)," Nat Meth 3, 793-796 (2006).
[CrossRef]

Zondervan, R.

R. Zondervan, F. Kulzer, M. A. Kolchenko, and M. Orrit, "Photobleaching of Rhodamine 6G in Poly(vinyl alcohol) at the ensemble and single-molecule levels," J. Phys. Chem. A 108, 1657-1665 (2004).
[CrossRef]

Biophys. J. (1)

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, "Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy," Biophys. J. 91, 4258-4272 (2006).
[CrossRef] [PubMed]

Chem. Phys. Lett. (1)

L. A. Deschenes and D. A. Vanden Bout, "Single molecule photobleaching: increasing photon yield and survival time through suppression of two-step photolysis," Chem. Phys. Lett. 365, 387-395 (2002).
[CrossRef]

J. Phys. Chem. A (2)

E. K. L. Yeow, S. M. Melnikov, T. D. M. Bell, F. C. DeSchryver, and J. Hofkens, "Characterizing the Fluorescence Intermittency and Photobleaching Kinetics of Dye Molecules Immobilized on a Glass Surface," J. Phys. Chem. A 110, 1726-1734 (2006).
[CrossRef] [PubMed]

R. Zondervan, F. Kulzer, M. A. Kolchenko, and M. Orrit, "Photobleaching of Rhodamine 6G in Poly(vinyl alcohol) at the ensemble and single-molecule levels," J. Phys. Chem. A 108, 1657-1665 (2004).
[CrossRef]

Meth. Cell. Biol. (1)

M. Osborn and K. Weber, "Immunofluorescence and Immunocytochemical Procedures with Affinity Purified Antibodies: Tubulin-Containing Structures," Meth. Cell. Biol. 24, 97-132 (1982).
[CrossRef]

Nat Meth (1)

M. J. Rust, M. Bates, and X. Zhuang, "Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)," Nat Meth 3, 793-796 (2006).
[CrossRef]

Nature (1)

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, "STED-microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis," Nature 440, 935 - 939 (2006).
[CrossRef] [PubMed]

Nature Methods (1)

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, "STED microscopy with continuous wave beams," Nature Methods 4, 915-918 (2007).
[CrossRef] [PubMed]

Opt. Express (2)

Opt. Lett. (4)

Phys. Rev. Lett. (1)

V. Westphal and S. W. Hell, "Nanoscale Resolution in the Focal Plane of an Optical Microscope," Phys. Rev. Lett. 94, 143903 (2005).
[CrossRef] [PubMed]

Proc. Nat. Acad. Sci. U.S.A. (1)

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, "Fluorescence microscopy with diffraction resolution limit broken by stimulated emission," Proc. Nat. Acad. Sci. U.S.A. 97, 8206-8210 (2000).
[CrossRef]

Proc. Natl. Acad. Sci. U. S. A. (1)

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell "Macromolecular-scale resolution in biological fluorescence microscopy," Proc. Natl. Acad. Sci. U. S. A. 103, 11440-11445 (2006).
[CrossRef] [PubMed]

Science (3)

Q1. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, "Imaging Intracellular Fluorescent Proteins at Nanometer Resolution," Science 313, 1642-1645 (2006).
[CrossRef] [PubMed]

S. W. Hell, "Far-Field Optical Nanoscopy," Science 316, 1153-1158 (2007).
[CrossRef] [PubMed]

P. Russell, "Photonic Crystal Fibers," Science 299, 358-362 (2003).
[CrossRef] [PubMed]

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Figures (3)

Fig. 1.
Fig. 1.

Experimental Setup. The randomly polarized laser beam is split into two beams using a polarizing beamsplitter cube (PB) from which the excitation and STED wavelengths are extracted by means of an interference filter (EF) and a prism-based wavelength selector (WS), respectively. Both beams are spatially filtered by coupling them into single-mode fibers (SMF), expanded and coupled into a confocal setup using two dichroic beamsplitters (D1, D2). The focal doughnut is created by passing the STED beam through a vortex phase mask (PM). DF: detection filter, MMF: multimode fiber; APD: avalanche photodiode; P: prism; S: slit.

Fig. 2.
Fig. 2.

Comparison between confocal (middle) and STED images (left) of randomly dispersed 40 nm fluorescent beads at the indicated wavelengths. The upper, center, and lower row show the data of red, crimson, and orange beads, respectively. Postprocessing the raw data (left) by a Richardson-Lucy deconvolution algorithm further enhances the details (STED+, right). The line profiles along the traces indicated by blue and purple arrows demonstrate that full-width-at-half-maximum values between 49 nm and 58 nm were achieved in the raw data. Scale bar: 500 nm.

Fig. 3.
Fig. 3.

Immunolabeled tubulin fibers imaged with an excitation wavelength of 570 nm (top), 630 nm (middle), and 532 nm (bottom). The comparison between the confocal reference image (left) and the STED image (right) reveals the gain in structural information obtained by STED; note that all images represent raw data. The line profiles along the traces indicated by the blue and purple arrows highlights details in the STED image (purple) that are not discerned by the confocal microscope (blue). Scale bar: 1 µm.

Tables (1)

Tables Icon

Table 1. Filter combinations used at different STED wavelengths.

Metrics