Abstract

We undertake a comprehensive study of the inverse square root dependence of spatial resolution on the saturation factor in stimulated emission depletion (STED) microscopy and generalize it to account for various focal depletion patterns. We used an experimental platform featuring a high quality depletion pattern which results in operation close to the optimal optical performance. Its superior image brightness and uniform effective resolution <25 nm are evidenced by imaging both isolated and self-organized convectively assembled fluorescent beads. For relevant saturation values, the generalized square-root law is shown to predict the practical resolution with high accuracy.

© 2008 Optical Society of America

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. S. G. Megason and S. E. Fraser, "Imaging in systems biology," Cell 130, 784-795 (2007).
    [CrossRef] [PubMed]
  2. S. W. Hell, "Far-Field Optical Nanoscopy," Science 316, 1153-1158 (2007).
    [CrossRef] [PubMed]
  3. W. E. Moerner, "New directions in single-molecule imaging and analysis," Proc. Natl. Acad. Sci. U. S. A. 104, 12596-12602 (2007).
    [CrossRef] [PubMed]
  4. A. Heinrichs, "Imaging: Overcoming the barrier," Nat. Rev. Mol. Cell Biol. 7, 707-707 (2006).
    [CrossRef]
  5. 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]
  6. T. A. Klar and S. W. Hell, "Subdiffraction resolution in far-field fluorescence microscopy," Opt. Lett. 24, 954-956 (1999).
    [CrossRef]
  7. M. Dyba and S. W. Hell, "Focal spots of size l/23 open up far-field fluorescence microscopy at 33 nm axial resolution," Phys. Rev. Lett. 88, 163901 (2002).
    [CrossRef] [PubMed]
  8. 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]
  9. 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]
  10. M. J. Rust, M. Bates, and X. Zhuang, "Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)," Nature Methods 3, 793-796 (2006).
    [CrossRef] [PubMed]
  11. A. Sharonov and R. M. Hochstrasser, "Wide-field subdiffraction imaging by accumulated binding of diffusing probes," Proc. Natl. Acad. Sci. U. S. A. 103, 18911-18916 (2006).
    [CrossRef] [PubMed]
  12. M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell "Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins," Proc. Natl. Acad. Sci. U. S. A. 102, 17565-17569 (2005).
    [CrossRef] [PubMed]
  13. M. G. L. Gustafsson, "Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution," Proc. Natl. Acad. Sci. U. S. A. 102, 13081-13086 (2005).
    [CrossRef] [PubMed]
  14. 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]
  15. R. J. Kittel, C. Wichmann, T. M. Rasse, W. Fouquet, M. Schmidt, A. Schmid, D. A. Wagh, C. Pawlu, R. Kellner, K. I. Willig, S. W. Hell, E. Buchner, M. Heckmann, and S. J. Sigrist, "Bruchpilot Promotes Active Zone Assembly, Ca2+-Channel Clustering, and Vesicle Release," Science 312, 1051-1054 (2006).
    [CrossRef] [PubMed]
  16. J. J. Sieber, K. I. Willig, C. Kutzner, C. Gerding-Reimers, B. Harke, G. Donnert, B. Rammner, C. Eggeling, S. W. Hell, H. Grubmüller, and T. Lang, "Anatomy and dynamics of a supramolecular membrane protein cluster," Science 317, 1072-1076 (2007).
    [CrossRef] [PubMed]
  17. B. Harke, C. K. Ullal, J. Keller, and S. W. Hell, "Three-Dimensional Nanoscopy of Colloidal Crystals," Nano. Lett. ASAP Article 10.1021/nl073164n (2008).
    [CrossRef]
  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. S. W. Hell, "Toward fluorescence nanoscopy," Nat. Biotechnol. 21, 1347-1355 (2003).
    [CrossRef] [PubMed]
  20. V. Westphal and S. W. Hell, "Nanoscale Resolution in the Focal Plane of an Optical Microscope," Phys. Rev. Lett. 94, 143903 (2005).
    [CrossRef] [PubMed]
  21. M. Dyba, J. Keller, and S. W. Hell, "Phase filter enhanced STED-4Pi fluorescence microscopy: theory and experiment," New J. Phys. 7 (2005).
    [CrossRef]
  22. 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]
  23. K. Willig, J. Keller, M. Bossi, and S. W. Hell, "STED microscopy resolves nanoparticle assemblies," New J. Phys. 8, 106 (2006).
    [CrossRef]
  24. N. D. Denkov, O. D. Velev, P. A. Kralchevsky, I. B. Ivanov, H. Yoshimura, and K. Nagayama, "Mechanism of Formation of 2-Dimensional Crystals from Latex-Particles on Substrates," Langmuir 8, 3183-3190 (1992).
    [CrossRef]
  25. V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, "Laser-diode-stimulated emission depletion microscopy," Appl. Phys. Lett. 82, 3125-3127 (2003).
    [CrossRef]

2007 (6)

S. G. Megason and S. E. Fraser, "Imaging in systems biology," Cell 130, 784-795 (2007).
[CrossRef] [PubMed]

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

W. E. Moerner, "New directions in single-molecule imaging and analysis," Proc. Natl. Acad. Sci. U. S. A. 104, 12596-12602 (2007).
[CrossRef] [PubMed]

J. J. Sieber, K. I. Willig, C. Kutzner, C. Gerding-Reimers, B. Harke, G. Donnert, B. Rammner, C. Eggeling, S. W. Hell, H. Grubmüller, and T. Lang, "Anatomy and dynamics of a supramolecular membrane protein cluster," Science 317, 1072-1076 (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]

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 (8)

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

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]

R. J. Kittel, C. Wichmann, T. M. Rasse, W. Fouquet, M. Schmidt, A. Schmid, D. A. Wagh, C. Pawlu, R. Kellner, K. I. Willig, S. W. Hell, E. Buchner, M. Heckmann, and S. J. Sigrist, "Bruchpilot Promotes Active Zone Assembly, Ca2+-Channel Clustering, and Vesicle Release," Science 312, 1051-1054 (2006).
[CrossRef] [PubMed]

A. Heinrichs, "Imaging: Overcoming the barrier," Nat. Rev. Mol. Cell Biol. 7, 707-707 (2006).
[CrossRef]

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. 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]

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

A. Sharonov and R. M. Hochstrasser, "Wide-field subdiffraction imaging by accumulated binding of diffusing probes," Proc. Natl. Acad. Sci. U. S. A. 103, 18911-18916 (2006).
[CrossRef] [PubMed]

2005 (4)

M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell "Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins," Proc. Natl. Acad. Sci. U. S. A. 102, 17565-17569 (2005).
[CrossRef] [PubMed]

M. G. L. Gustafsson, "Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution," Proc. Natl. Acad. Sci. U. S. A. 102, 13081-13086 (2005).
[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]

M. Dyba, J. Keller, and S. W. Hell, "Phase filter enhanced STED-4Pi fluorescence microscopy: theory and experiment," New J. Phys. 7 (2005).
[CrossRef]

2003 (2)

S. W. Hell, "Toward fluorescence nanoscopy," Nat. Biotechnol. 21, 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, 3125-3127 (2003).
[CrossRef]

2002 (1)

M. Dyba and S. W. Hell, "Focal spots of size l/23 open up far-field fluorescence microscopy at 33 nm axial resolution," Phys. Rev. Lett. 88, 163901 (2002).
[CrossRef] [PubMed]

1999 (1)

1994 (1)

1992 (1)

N. D. Denkov, O. D. Velev, P. A. Kralchevsky, I. B. Ivanov, H. Yoshimura, and K. Nagayama, "Mechanism of Formation of 2-Dimensional Crystals from Latex-Particles on Substrates," Langmuir 8, 3183-3190 (1992).
[CrossRef]

Bates, M.

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

Betzig, E.

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]

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, 3125-3127 (2003).
[CrossRef]

Bonifacino, J. S.

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]

Bossi, M.

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

Buchner, E.

R. J. Kittel, C. Wichmann, T. M. Rasse, W. Fouquet, M. Schmidt, A. Schmid, D. A. Wagh, C. Pawlu, R. Kellner, K. I. Willig, S. W. Hell, E. Buchner, M. Heckmann, and S. J. Sigrist, "Bruchpilot Promotes Active Zone Assembly, Ca2+-Channel Clustering, and Vesicle Release," Science 312, 1051-1054 (2006).
[CrossRef] [PubMed]

Davidson, M. W.

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]

Denkov, N. D.

N. D. Denkov, O. D. Velev, P. A. Kralchevsky, I. B. Ivanov, H. Yoshimura, and K. Nagayama, "Mechanism of Formation of 2-Dimensional Crystals from Latex-Particles on Substrates," Langmuir 8, 3183-3190 (1992).
[CrossRef]

Donnert, G.

J. J. Sieber, K. I. Willig, C. Kutzner, C. Gerding-Reimers, B. Harke, G. Donnert, B. Rammner, C. Eggeling, S. W. Hell, H. Grubmüller, and T. Lang, "Anatomy and dynamics of a supramolecular membrane protein cluster," Science 317, 1072-1076 (2007).
[CrossRef] [PubMed]

Dyba, M.

M. Dyba, J. Keller, and S. W. Hell, "Phase filter enhanced STED-4Pi fluorescence microscopy: theory and experiment," New J. Phys. 7 (2005).
[CrossRef]

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

M. Dyba and S. W. Hell, "Focal spots of size l/23 open up far-field fluorescence microscopy at 33 nm axial resolution," Phys. Rev. Lett. 88, 163901 (2002).
[CrossRef] [PubMed]

Eggeling, C.

J. J. Sieber, K. I. Willig, C. Kutzner, C. Gerding-Reimers, B. Harke, G. Donnert, B. Rammner, C. Eggeling, S. W. Hell, H. Grubmüller, and T. Lang, "Anatomy and dynamics of a supramolecular membrane protein cluster," Science 317, 1072-1076 (2007).
[CrossRef] [PubMed]

M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell "Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins," Proc. Natl. Acad. Sci. U. S. A. 102, 17565-17569 (2005).
[CrossRef] [PubMed]

Fouquet, W.

R. J. Kittel, C. Wichmann, T. M. Rasse, W. Fouquet, M. Schmidt, A. Schmid, D. A. Wagh, C. Pawlu, R. Kellner, K. I. Willig, S. W. Hell, E. Buchner, M. Heckmann, and S. J. Sigrist, "Bruchpilot Promotes Active Zone Assembly, Ca2+-Channel Clustering, and Vesicle Release," Science 312, 1051-1054 (2006).
[CrossRef] [PubMed]

Fraser, S. E.

S. G. Megason and S. E. Fraser, "Imaging in systems biology," Cell 130, 784-795 (2007).
[CrossRef] [PubMed]

Gerding-Reimers, C.

J. J. Sieber, K. I. Willig, C. Kutzner, C. Gerding-Reimers, B. Harke, G. Donnert, B. Rammner, C. Eggeling, S. W. Hell, H. Grubmüller, and T. Lang, "Anatomy and dynamics of a supramolecular membrane protein cluster," Science 317, 1072-1076 (2007).
[CrossRef] [PubMed]

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]

Grubmüller, H.

J. J. Sieber, K. I. Willig, C. Kutzner, C. Gerding-Reimers, B. Harke, G. Donnert, B. Rammner, C. Eggeling, S. W. Hell, H. Grubmüller, and T. Lang, "Anatomy and dynamics of a supramolecular membrane protein cluster," Science 317, 1072-1076 (2007).
[CrossRef] [PubMed]

Gustafsson, M. G. L.

M. G. L. Gustafsson, "Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution," Proc. Natl. Acad. Sci. U. S. A. 102, 13081-13086 (2005).
[CrossRef] [PubMed]

Harke, B.

J. J. Sieber, K. I. Willig, C. Kutzner, C. Gerding-Reimers, B. Harke, G. Donnert, B. Rammner, C. Eggeling, S. W. Hell, H. Grubmüller, and T. Lang, "Anatomy and dynamics of a supramolecular membrane protein cluster," Science 317, 1072-1076 (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]

Heckmann, M.

R. J. Kittel, C. Wichmann, T. M. Rasse, W. Fouquet, M. Schmidt, A. Schmid, D. A. Wagh, C. Pawlu, R. Kellner, K. I. Willig, S. W. Hell, E. Buchner, M. Heckmann, and S. J. Sigrist, "Bruchpilot Promotes Active Zone Assembly, Ca2+-Channel Clustering, and Vesicle Release," Science 312, 1051-1054 (2006).
[CrossRef] [PubMed]

Heinrichs, A.

A. Heinrichs, "Imaging: Overcoming the barrier," Nat. Rev. Mol. Cell Biol. 7, 707-707 (2006).
[CrossRef]

Hell, S. W.

S. W. Hell, "Far-Field Optical Nanoscopy," Science 316, 1153-1158 (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]

J. J. Sieber, K. I. Willig, C. Kutzner, C. Gerding-Reimers, B. Harke, G. Donnert, B. Rammner, C. Eggeling, S. W. Hell, H. Grubmüller, and T. Lang, "Anatomy and dynamics of a supramolecular membrane protein cluster," Science 317, 1072-1076 (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]

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

R. J. Kittel, C. Wichmann, T. M. Rasse, W. Fouquet, M. Schmidt, A. Schmid, D. A. Wagh, C. Pawlu, R. Kellner, K. I. Willig, S. W. Hell, E. Buchner, M. Heckmann, and S. J. Sigrist, "Bruchpilot Promotes Active Zone Assembly, Ca2+-Channel Clustering, and Vesicle Release," Science 312, 1051-1054 (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]

M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell "Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins," Proc. Natl. Acad. Sci. U. S. A. 102, 17565-17569 (2005).
[CrossRef] [PubMed]

M. Dyba, J. Keller, and S. W. Hell, "Phase filter enhanced STED-4Pi fluorescence microscopy: theory and experiment," New J. Phys. 7 (2005).
[CrossRef]

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

S. W. Hell, "Toward fluorescence nanoscopy," Nat. Biotechnol. 21, 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, 3125-3127 (2003).
[CrossRef]

M. Dyba and S. W. Hell, "Focal spots of size l/23 open up far-field fluorescence microscopy at 33 nm axial resolution," Phys. Rev. Lett. 88, 163901 (2002).
[CrossRef] [PubMed]

T. A. Klar and S. W. Hell, "Subdiffraction resolution in far-field fluorescence microscopy," Opt. Lett. 24, 954-956 (1999).
[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.

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]

Hochstrasser, R. M.

A. Sharonov and R. M. Hochstrasser, "Wide-field subdiffraction imaging by accumulated binding of diffusing probes," Proc. Natl. Acad. Sci. U. S. A. 103, 18911-18916 (2006).
[CrossRef] [PubMed]

Hofmann, M.

M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell "Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins," Proc. Natl. Acad. Sci. U. S. A. 102, 17565-17569 (2005).
[CrossRef] [PubMed]

Ivanov, I. B.

N. D. Denkov, O. D. Velev, P. A. Kralchevsky, I. B. Ivanov, H. Yoshimura, and K. Nagayama, "Mechanism of Formation of 2-Dimensional Crystals from Latex-Particles on Substrates," Langmuir 8, 3183-3190 (1992).
[CrossRef]

Jahn, R.

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.

M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell "Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins," Proc. Natl. Acad. Sci. U. S. A. 102, 17565-17569 (2005).
[CrossRef] [PubMed]

Kastrup, L.

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

Keller, J.

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

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

M. Dyba, J. Keller, and S. W. Hell, "Phase filter enhanced STED-4Pi fluorescence microscopy: theory and experiment," New J. Phys. 7 (2005).
[CrossRef]

Kellner, R.

R. J. Kittel, C. Wichmann, T. M. Rasse, W. Fouquet, M. Schmidt, A. Schmid, D. A. Wagh, C. Pawlu, R. Kellner, K. I. Willig, S. W. Hell, E. Buchner, M. Heckmann, and S. J. Sigrist, "Bruchpilot Promotes Active Zone Assembly, Ca2+-Channel Clustering, and Vesicle Release," Science 312, 1051-1054 (2006).
[CrossRef] [PubMed]

Kittel, R. J.

R. J. Kittel, C. Wichmann, T. M. Rasse, W. Fouquet, M. Schmidt, A. Schmid, D. A. Wagh, C. Pawlu, R. Kellner, K. I. Willig, S. W. Hell, E. Buchner, M. Heckmann, and S. J. Sigrist, "Bruchpilot Promotes Active Zone Assembly, Ca2+-Channel Clustering, and Vesicle Release," Science 312, 1051-1054 (2006).
[CrossRef] [PubMed]

Klar, T. A.

Kralchevsky, P. A.

N. D. Denkov, O. D. Velev, P. A. Kralchevsky, I. B. Ivanov, H. Yoshimura, and K. Nagayama, "Mechanism of Formation of 2-Dimensional Crystals from Latex-Particles on Substrates," Langmuir 8, 3183-3190 (1992).
[CrossRef]

Kutzner, C.

J. J. Sieber, K. I. Willig, C. Kutzner, C. Gerding-Reimers, B. Harke, G. Donnert, B. Rammner, C. Eggeling, S. W. Hell, H. Grubmüller, and T. Lang, "Anatomy and dynamics of a supramolecular membrane protein cluster," Science 317, 1072-1076 (2007).
[CrossRef] [PubMed]

Lang, T.

J. J. Sieber, K. I. Willig, C. Kutzner, C. Gerding-Reimers, B. Harke, G. Donnert, B. Rammner, C. Eggeling, S. W. Hell, H. Grubmüller, and T. Lang, "Anatomy and dynamics of a supramolecular membrane protein cluster," Science 317, 1072-1076 (2007).
[CrossRef] [PubMed]

Lindwasser, O. W.

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.

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]

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]

Megason, S. G.

S. G. Megason and S. E. Fraser, "Imaging in systems biology," Cell 130, 784-795 (2007).
[CrossRef] [PubMed]

Moerner, W. E.

W. E. Moerner, "New directions in single-molecule imaging and analysis," Proc. Natl. Acad. Sci. U. S. A. 104, 12596-12602 (2007).
[CrossRef] [PubMed]

Nagayama, K.

N. D. Denkov, O. D. Velev, P. A. Kralchevsky, I. B. Ivanov, H. Yoshimura, and K. Nagayama, "Mechanism of Formation of 2-Dimensional Crystals from Latex-Particles on Substrates," Langmuir 8, 3183-3190 (1992).
[CrossRef]

Olenych, S.

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]

Patterson, G. H.

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]

Pawlu, C.

R. J. Kittel, C. Wichmann, T. M. Rasse, W. Fouquet, M. Schmidt, A. Schmid, D. A. Wagh, C. Pawlu, R. Kellner, K. I. Willig, S. W. Hell, E. Buchner, M. Heckmann, and S. J. Sigrist, "Bruchpilot Promotes Active Zone Assembly, Ca2+-Channel Clustering, and Vesicle Release," Science 312, 1051-1054 (2006).
[CrossRef] [PubMed]

Rammner, B.

J. J. Sieber, K. I. Willig, C. Kutzner, C. Gerding-Reimers, B. Harke, G. Donnert, B. Rammner, C. Eggeling, S. W. Hell, H. Grubmüller, and T. Lang, "Anatomy and dynamics of a supramolecular membrane protein cluster," Science 317, 1072-1076 (2007).
[CrossRef] [PubMed]

Rasse, T. M.

R. J. Kittel, C. Wichmann, T. M. Rasse, W. Fouquet, M. Schmidt, A. Schmid, D. A. Wagh, C. Pawlu, R. Kellner, K. I. Willig, S. W. Hell, E. Buchner, M. Heckmann, and S. J. Sigrist, "Bruchpilot Promotes Active Zone Assembly, Ca2+-Channel Clustering, and Vesicle Release," Science 312, 1051-1054 (2006).
[CrossRef] [PubMed]

Rizzoli, S. O.

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]

Rust, M. J.

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

Schmid, A.

R. J. Kittel, C. Wichmann, T. M. Rasse, W. Fouquet, M. Schmidt, A. Schmid, D. A. Wagh, C. Pawlu, R. Kellner, K. I. Willig, S. W. Hell, E. Buchner, M. Heckmann, and S. J. Sigrist, "Bruchpilot Promotes Active Zone Assembly, Ca2+-Channel Clustering, and Vesicle Release," Science 312, 1051-1054 (2006).
[CrossRef] [PubMed]

Schmidt, M.

R. J. Kittel, C. Wichmann, T. M. Rasse, W. Fouquet, M. Schmidt, A. Schmid, D. A. Wagh, C. Pawlu, R. Kellner, K. I. Willig, S. W. Hell, E. Buchner, M. Heckmann, and S. J. Sigrist, "Bruchpilot Promotes Active Zone Assembly, Ca2+-Channel Clustering, and Vesicle Release," Science 312, 1051-1054 (2006).
[CrossRef] [PubMed]

Schönle, A.

Sharonov, A.

A. Sharonov and R. M. Hochstrasser, "Wide-field subdiffraction imaging by accumulated binding of diffusing probes," Proc. Natl. Acad. Sci. U. S. A. 103, 18911-18916 (2006).
[CrossRef] [PubMed]

Sieber, J. J.

J. J. Sieber, K. I. Willig, C. Kutzner, C. Gerding-Reimers, B. Harke, G. Donnert, B. Rammner, C. Eggeling, S. W. Hell, H. Grubmüller, and T. Lang, "Anatomy and dynamics of a supramolecular membrane protein cluster," Science 317, 1072-1076 (2007).
[CrossRef] [PubMed]

Sigrist, S. J.

R. J. Kittel, C. Wichmann, T. M. Rasse, W. Fouquet, M. Schmidt, A. Schmid, D. A. Wagh, C. Pawlu, R. Kellner, K. I. Willig, S. W. Hell, E. Buchner, M. Heckmann, and S. J. Sigrist, "Bruchpilot Promotes Active Zone Assembly, Ca2+-Channel Clustering, and Vesicle Release," Science 312, 1051-1054 (2006).
[CrossRef] [PubMed]

Sougrat, R.

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]

Velev, O. D.

N. D. Denkov, O. D. Velev, P. A. Kralchevsky, I. B. Ivanov, H. Yoshimura, and K. Nagayama, "Mechanism of Formation of 2-Dimensional Crystals from Latex-Particles on Substrates," Langmuir 8, 3183-3190 (1992).
[CrossRef]

Wagh, D. A.

R. J. Kittel, C. Wichmann, T. M. Rasse, W. Fouquet, M. Schmidt, A. Schmid, D. A. Wagh, C. Pawlu, R. Kellner, K. I. Willig, S. W. Hell, E. Buchner, M. Heckmann, and S. J. Sigrist, "Bruchpilot Promotes Active Zone Assembly, Ca2+-Channel Clustering, and Vesicle Release," Science 312, 1051-1054 (2006).
[CrossRef] [PubMed]

Westphal, V.

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]

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

Wichmann, C.

R. J. Kittel, C. Wichmann, T. M. Rasse, W. Fouquet, M. Schmidt, A. Schmid, D. A. Wagh, C. Pawlu, R. Kellner, K. I. Willig, S. W. Hell, E. Buchner, M. Heckmann, and S. J. Sigrist, "Bruchpilot Promotes Active Zone Assembly, Ca2+-Channel Clustering, and Vesicle Release," Science 312, 1051-1054 (2006).
[CrossRef] [PubMed]

Wichmann, J.

Willig, K.

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

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]

J. J. Sieber, K. I. Willig, C. Kutzner, C. Gerding-Reimers, B. Harke, G. Donnert, B. Rammner, C. Eggeling, S. W. Hell, H. Grubmüller, and T. Lang, "Anatomy and dynamics of a supramolecular membrane protein cluster," Science 317, 1072-1076 (2007).
[CrossRef] [PubMed]

R. J. Kittel, C. Wichmann, T. M. Rasse, W. Fouquet, M. Schmidt, A. Schmid, D. A. Wagh, C. Pawlu, R. Kellner, K. I. Willig, S. W. Hell, E. Buchner, M. Heckmann, and S. J. Sigrist, "Bruchpilot Promotes Active Zone Assembly, Ca2+-Channel Clustering, and Vesicle Release," Science 312, 1051-1054 (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]

Yoshimura, H.

N. D. Denkov, O. D. Velev, P. A. Kralchevsky, I. B. Ivanov, H. Yoshimura, and K. Nagayama, "Mechanism of Formation of 2-Dimensional Crystals from Latex-Particles on Substrates," Langmuir 8, 3183-3190 (1992).
[CrossRef]

Zhuang, X.

M. J. Rust, M. Bates, and X. Zhuang, "Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)," Nature Methods 3, 793-796 (2006).
[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, 3125-3127 (2003).
[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]

Cell (1)

S. G. Megason and S. E. Fraser, "Imaging in systems biology," Cell 130, 784-795 (2007).
[CrossRef] [PubMed]

Langmuir (1)

N. D. Denkov, O. D. Velev, P. A. Kralchevsky, I. B. Ivanov, H. Yoshimura, and K. Nagayama, "Mechanism of Formation of 2-Dimensional Crystals from Latex-Particles on Substrates," Langmuir 8, 3183-3190 (1992).
[CrossRef]

Nat. Biotechnol. (1)

S. W. Hell, "Toward fluorescence nanoscopy," Nat. Biotechnol. 21, 1347-1355 (2003).
[CrossRef] [PubMed]

Nat. Rev. Mol. Cell Biol. (1)

A. Heinrichs, "Imaging: Overcoming the barrier," Nat. Rev. Mol. Cell Biol. 7, 707-707 (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 (2)

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

New J. Phys. (2)

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

M. Dyba, J. Keller, and S. W. Hell, "Phase filter enhanced STED-4Pi fluorescence microscopy: theory and experiment," New J. Phys. 7 (2005).
[CrossRef]

Opt. Express (1)

Opt. Lett. (2)

Phys. Rev. Lett. (2)

M. Dyba and S. W. Hell, "Focal spots of size l/23 open up far-field fluorescence microscopy at 33 nm axial resolution," Phys. Rev. Lett. 88, 163901 (2002).
[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]

Proc. Natl. Acad. Sci. U. S. A. (4)

A. Sharonov and R. M. Hochstrasser, "Wide-field subdiffraction imaging by accumulated binding of diffusing probes," Proc. Natl. Acad. Sci. U. S. A. 103, 18911-18916 (2006).
[CrossRef] [PubMed]

M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell "Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins," Proc. Natl. Acad. Sci. U. S. A. 102, 17565-17569 (2005).
[CrossRef] [PubMed]

M. G. L. Gustafsson, "Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution," Proc. Natl. Acad. Sci. U. S. A. 102, 13081-13086 (2005).
[CrossRef] [PubMed]

W. E. Moerner, "New directions in single-molecule imaging and analysis," Proc. Natl. Acad. Sci. U. S. A. 104, 12596-12602 (2007).
[CrossRef] [PubMed]

Science (4)

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

R. J. Kittel, C. Wichmann, T. M. Rasse, W. Fouquet, M. Schmidt, A. Schmid, D. A. Wagh, C. Pawlu, R. Kellner, K. I. Willig, S. W. Hell, E. Buchner, M. Heckmann, and S. J. Sigrist, "Bruchpilot Promotes Active Zone Assembly, Ca2+-Channel Clustering, and Vesicle Release," Science 312, 1051-1054 (2006).
[CrossRef] [PubMed]

J. J. Sieber, K. I. Willig, C. Kutzner, C. Gerding-Reimers, B. Harke, G. Donnert, B. Rammner, C. Eggeling, S. W. Hell, H. Grubmüller, and T. Lang, "Anatomy and dynamics of a supramolecular membrane protein cluster," Science 317, 1072-1076 (2007).
[CrossRef] [PubMed]

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]

Other (1)

B. Harke, C. K. Ullal, J. Keller, and S. W. Hell, "Three-Dimensional Nanoscopy of Colloidal Crystals," Nano. Lett. ASAP Article 10.1021/nl073164n (2008).
[CrossRef]

Supplementary Material (2)

» Media 1: AVI (2823 KB)     
» Media 2: AVI (3826 KB)     

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 (5)

Fig. 1.
Fig. 1.

Experimental platform; (a) Setup. PMF: polarisation maintaining fibre; APD: avalanche photo diode; PH: pinhole; TL: tube lens; DM: dichroic mirror; OL: objective lens; SF6: glass rods. (b) Absorption and emission spectrum of a solution of 24 nm fluorescent beads used in the presented measurements. (c) 3D view of the phase masks used for the measurements.

Fig. 2.
Fig. 2.

Resolution enhancement realized by STED microscopy. (a,b) Confocal(a) and STED (b) image of 24 nm fluorescent beads on a cover slip. (c–g) The area of the white rectangle shown in (a) and (b) recorded with different STED intensities. The resolution gain can directly be observed. (h) STED depletion measured on the same sample. The intensity settings for the measurements (c–g) are marked by red arrows. Scale bar in (a,b) 1 µm, in (c–g) 200 nm. Images were recorded with a pixel size of 15 nm and then interpolated to a pixel size of 10 nm. Multimedia file movie 1(avi, 2.6 MB).

Fig. 3.
Fig. 3.

Resolution dependence on the saturation factor for different depletion patterns.(a,d) Parabolic fit (red dashed line) to the depletion pattern for the lateral (a) and axial (d) direction. (b,e) Experimental data for lateral (b) and axial (e) confinements. Note the small error bars. (c,f) The simulated values for the lateral (c) and axial (f) resolution for different saturation factors. For both the simulated and experimental values an excellent match to the square root dependence (red dotted line) is observed.

Fig. 4.
Fig. 4.

Relative peak intensity of the STED image versus the factor of resolution enhancement in comparison to a confocal microscope. The value of 100 % implies that the fluorescence from the doughnut centre keeps the same value as without the STED beam. With larger resolution enhancement factors, the non-zero central minimum of the doughnut reduces the desired fluorescence signal from the doughnut centre. Nonetheless, even for a factor of 10 resolution increase in all directions in the focal plane, the percentage of photons discarded by STED is <25 %.

Fig. 5.
Fig. 5.

Imaging of 200 nm self assembled colloidals: (a) confocal image with the STED counterpart. (b). Scale bar 1µm, in insets scale bar 250 nm (movie2, avi, 2.8 MB). All images are raw data.

Equations (8)

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

Δ x λ n ς
ς = I STED I s
η ( x ) exp ( ln ( 2 ) I STED ( x ) I S )
I STED ( x ) = 4 I STED a 2 x 2
h eff ( x ) = h c ( x ) η ( x )
h eff ( x ) = exp ( 4 ln ( 2 ) x 2 ( d c 2 + a 2 ς ) )
d = d c 1 + d c 2 a 2 ς
d 1 ( a ς )

Metrics