Abstract

We present a simplified single-beam scheme for depletion-based sub-diffraction-limited imaging which allows for less restrictive illumination conditions. This is done by introducing the concept of fluorophores exhibiting emission depletion upon saturation. We discuss the circumstances under which such a depletion based process is possible, and derive the scaling of the spatial resolution utilizing this scheme. Next, we analyze the proper illumination conditions both in space and time required for sub diffraction limited imaging, and show that it is applicable only under pulsed excitation. Finally, our scheme’s advantages and shortcomings relative to alternative realizations of depletion-based sub-diffraction-limited microscopy are discussed.

©2009 Optical Society of America

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References

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  1. S. W. Hell, “Far field optical nanoscopy,” Science 316, 1153–1158 (2007).
    [Crossref] [PubMed]
  2. R. W. Boyd, “Nonlinear Optics,” second edition, (Academic Press, 2003).
  3. M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3, No. 10, 793–796 (2006).
    [Crossref] [PubMed]
  4. M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317, 1749–1753 (2007).
    [Crossref] [PubMed]
  5. 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. 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]
  7. A. Sharonov and R. M. Hochstrasser, “Wide-Field Subdiffraction Imaging by Accumulated Binding of Diffusing Probes,” Proc. Natl. Acad. Sci. 103, No. 50, 18911–18916 (2006).
    [Crossref] [PubMed]
  8. H. Bock, C. Geisler, C. A. Wurm, C. von Middendorff, S. Jakobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-Color Far-Field Fluorescence Nanoscopy Based on Photoswitchable Emitters,” Appl. Phys. B 88, 161–165 (2007).
    [Crossref]
  9. A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schonle, and S. W. Hell, “Fluorescence Nanoscopy in Whole Cells by Asynchronous Localization of Photoswitching Emitters,” Biophys. J. 93 No. 11, 3285–3290 (2007).
    [Crossref] [PubMed]
  10. S. W. Hell and J. Wichmann, “Breaking the diffraction limit by stimulated emission: Stimulated emission sincedepletion microscopy,” Opt. Lett. 19780–782, (1994).
    [Crossref] [PubMed]
  11. K. I. Willing, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuose wave beams,” Nat. Methods 4,No. 11, 915–918 (2007).
    [Crossref]
  12. S. E. Irvine, T. Staudt, E. Rittweger, J. Engelhardt, and S. W. Hell, “Direct Light-Driven Modulation of Luminescence from Mn-Doped ZnSe Quantum Dots,” Angew. Chem. Int. Ed. 47, Vol. 14, 2685–2688 (2008).
    [Crossref]
  13. S. Bretschneider, C. Eggeling, and S. W. Hell, “Breaking the Diffraction Barrier in Fluorescence Microscopy by Optical Shelving,” Phys. Rev. Lett. 98, 218103(1–4) (2007).
    [Crossref] [PubMed]
  14. G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92, L67–L69 (2007).
    [Crossref] [PubMed]
  15. S. W. Hell, “Strategy for far-field optical imaging and writing without diffraction limit,” Phys. Lett. A. 326, 140–145 (2004).
    [Crossref]
  16. B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schonle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16, 4154–4162 (2008).
    [Crossref] [PubMed]
  17. M. Bates, T. R. Blosser, and X. Zhuang, “Short-Range Spectroscopic Ruler Based on a Single-Molecule Optical Switch,” Phys. Rev. Lett. 94, 108101(1–4) (2005).
    [Crossref] [PubMed]
  18. M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317, 1749–1753 (2007).
    [Crossref] [PubMed]
  19. G. H. Patterson and J. Lippincott-Schwartz, “A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells,” Science 297, 1873–1877 (2002).
    [Crossref] [PubMed]

2008 (2)

S. E. Irvine, T. Staudt, E. Rittweger, J. Engelhardt, and S. W. Hell, “Direct Light-Driven Modulation of Luminescence from Mn-Doped ZnSe Quantum Dots,” Angew. Chem. Int. Ed. 47, Vol. 14, 2685–2688 (2008).
[Crossref]

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

2007 (8)

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317, 1749–1753 (2007).
[Crossref] [PubMed]

S. Bretschneider, C. Eggeling, and S. W. Hell, “Breaking the Diffraction Barrier in Fluorescence Microscopy by Optical Shelving,” Phys. Rev. Lett. 98, 218103(1–4) (2007).
[Crossref] [PubMed]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92, L67–L69 (2007).
[Crossref] [PubMed]

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317, 1749–1753 (2007).
[Crossref] [PubMed]

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

H. Bock, C. Geisler, C. A. Wurm, C. von Middendorff, S. Jakobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-Color Far-Field Fluorescence Nanoscopy Based on Photoswitchable Emitters,” Appl. Phys. B 88, 161–165 (2007).
[Crossref]

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schonle, and S. W. Hell, “Fluorescence Nanoscopy in Whole Cells by Asynchronous Localization of Photoswitching Emitters,” Biophys. J. 93 No. 11, 3285–3290 (2007).
[Crossref] [PubMed]

K. I. Willing, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuose wave beams,” Nat. Methods 4,No. 11, 915–918 (2007).
[Crossref]

2006 (4)

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3, No. 10, 793–796 (2006).
[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]

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]

A. Sharonov and R. M. Hochstrasser, “Wide-Field Subdiffraction Imaging by Accumulated Binding of Diffusing Probes,” Proc. Natl. Acad. Sci. 103, No. 50, 18911–18916 (2006).
[Crossref] [PubMed]

2005 (1)

M. Bates, T. R. Blosser, and X. Zhuang, “Short-Range Spectroscopic Ruler Based on a Single-Molecule Optical Switch,” Phys. Rev. Lett. 94, 108101(1–4) (2005).
[Crossref] [PubMed]

2004 (1)

S. W. Hell, “Strategy for far-field optical imaging and writing without diffraction limit,” Phys. Lett. A. 326, 140–145 (2004).
[Crossref]

2002 (1)

G. H. Patterson and J. Lippincott-Schwartz, “A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells,” Science 297, 1873–1877 (2002).
[Crossref] [PubMed]

1994 (1)

Andresen, M.

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schonle, and S. W. Hell, “Fluorescence Nanoscopy in Whole Cells by Asynchronous Localization of Photoswitching Emitters,” Biophys. J. 93 No. 11, 3285–3290 (2007).
[Crossref] [PubMed]

Bates, M.

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317, 1749–1753 (2007).
[Crossref] [PubMed]

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317, 1749–1753 (2007).
[Crossref] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3, No. 10, 793–796 (2006).
[Crossref] [PubMed]

M. Bates, T. R. Blosser, and X. Zhuang, “Short-Range Spectroscopic Ruler Based on a Single-Molecule Optical Switch,” Phys. Rev. Lett. 94, 108101(1–4) (2005).
[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]

Blosser, T. R.

M. Bates, T. R. Blosser, and X. Zhuang, “Short-Range Spectroscopic Ruler Based on a Single-Molecule Optical Switch,” Phys. Rev. Lett. 94, 108101(1–4) (2005).
[Crossref] [PubMed]

Bock, H.

H. Bock, C. Geisler, C. A. Wurm, C. von Middendorff, S. Jakobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-Color Far-Field Fluorescence Nanoscopy Based on Photoswitchable Emitters,” Appl. Phys. B 88, 161–165 (2007).
[Crossref]

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schonle, and S. W. Hell, “Fluorescence Nanoscopy in Whole Cells by Asynchronous Localization of Photoswitching Emitters,” Biophys. J. 93 No. 11, 3285–3290 (2007).
[Crossref] [PubMed]

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]

Boyd, R. W.

R. W. Boyd, “Nonlinear Optics,” second edition, (Academic Press, 2003).

Bretschneider, S.

S. Bretschneider, C. Eggeling, and S. W. Hell, “Breaking the Diffraction Barrier in Fluorescence Microscopy by Optical Shelving,” Phys. Rev. Lett. 98, 218103(1–4) (2007).
[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]

Dempsey, G. T.

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317, 1749–1753 (2007).
[Crossref] [PubMed]

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317, 1749–1753 (2007).
[Crossref] [PubMed]

Donnert, G.

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92, L67–L69 (2007).
[Crossref] [PubMed]

Eggeling, C.

S. Bretschneider, C. Eggeling, and S. W. Hell, “Breaking the Diffraction Barrier in Fluorescence Microscopy by Optical Shelving,” Phys. Rev. Lett. 98, 218103(1–4) (2007).
[Crossref] [PubMed]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92, L67–L69 (2007).
[Crossref] [PubMed]

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schonle, and S. W. Hell, “Fluorescence Nanoscopy in Whole Cells by Asynchronous Localization of Photoswitching Emitters,” Biophys. J. 93 No. 11, 3285–3290 (2007).
[Crossref] [PubMed]

H. Bock, C. Geisler, C. A. Wurm, C. von Middendorff, S. Jakobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-Color Far-Field Fluorescence Nanoscopy Based on Photoswitchable Emitters,” Appl. Phys. B 88, 161–165 (2007).
[Crossref]

Egner, A.

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schonle, and S. W. Hell, “Fluorescence Nanoscopy in Whole Cells by Asynchronous Localization of Photoswitching Emitters,” Biophys. J. 93 No. 11, 3285–3290 (2007).
[Crossref] [PubMed]

H. Bock, C. Geisler, C. A. Wurm, C. von Middendorff, S. Jakobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-Color Far-Field Fluorescence Nanoscopy Based on Photoswitchable Emitters,” Appl. Phys. B 88, 161–165 (2007).
[Crossref]

Engelhardt, J.

S. E. Irvine, T. Staudt, E. Rittweger, J. Engelhardt, and S. W. Hell, “Direct Light-Driven Modulation of Luminescence from Mn-Doped ZnSe Quantum Dots,” Angew. Chem. Int. Ed. 47, Vol. 14, 2685–2688 (2008).
[Crossref]

Geisler, C.

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schonle, and S. W. Hell, “Fluorescence Nanoscopy in Whole Cells by Asynchronous Localization of Photoswitching Emitters,” Biophys. J. 93 No. 11, 3285–3290 (2007).
[Crossref] [PubMed]

H. Bock, C. Geisler, C. A. Wurm, C. von Middendorff, S. Jakobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-Color Far-Field Fluorescence Nanoscopy Based on Photoswitchable Emitters,” Appl. Phys. B 88, 161–165 (2007).
[Crossref]

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]

Harke, B.

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

K. I. Willing, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuose wave beams,” Nat. Methods 4,No. 11, 915–918 (2007).
[Crossref]

Hell, S. W.

S. E. Irvine, T. Staudt, E. Rittweger, J. Engelhardt, and S. W. Hell, “Direct Light-Driven Modulation of Luminescence from Mn-Doped ZnSe Quantum Dots,” Angew. Chem. Int. Ed. 47, Vol. 14, 2685–2688 (2008).
[Crossref]

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

S. Bretschneider, C. Eggeling, and S. W. Hell, “Breaking the Diffraction Barrier in Fluorescence Microscopy by Optical Shelving,” Phys. Rev. Lett. 98, 218103(1–4) (2007).
[Crossref] [PubMed]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92, L67–L69 (2007).
[Crossref] [PubMed]

K. I. Willing, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuose wave beams,” Nat. Methods 4,No. 11, 915–918 (2007).
[Crossref]

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schonle, and S. W. Hell, “Fluorescence Nanoscopy in Whole Cells by Asynchronous Localization of Photoswitching Emitters,” Biophys. J. 93 No. 11, 3285–3290 (2007).
[Crossref] [PubMed]

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

H. Bock, C. Geisler, C. A. Wurm, C. von Middendorff, S. Jakobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-Color Far-Field Fluorescence Nanoscopy Based on Photoswitchable Emitters,” Appl. Phys. B 88, 161–165 (2007).
[Crossref]

S. W. Hell, “Strategy for far-field optical imaging and writing without diffraction limit,” Phys. Lett. A. 326, 140–145 (2004).
[Crossref]

S. W. Hell and J. Wichmann, “Breaking the diffraction limit by stimulated emission: Stimulated emission sincedepletion microscopy,” Opt. Lett. 19780–782, (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. 103, No. 50, 18911–18916 (2006).
[Crossref] [PubMed]

Huang, B.

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317, 1749–1753 (2007).
[Crossref] [PubMed]

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317, 1749–1753 (2007).
[Crossref] [PubMed]

Irvine, S. E.

S. E. Irvine, T. Staudt, E. Rittweger, J. Engelhardt, and S. W. Hell, “Direct Light-Driven Modulation of Luminescence from Mn-Doped ZnSe Quantum Dots,” Angew. Chem. Int. Ed. 47, Vol. 14, 2685–2688 (2008).
[Crossref]

Jahn, R.

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92, L67–L69 (2007).
[Crossref] [PubMed]

Jakobs, S.

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92, L67–L69 (2007).
[Crossref] [PubMed]

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schonle, and S. W. Hell, “Fluorescence Nanoscopy in Whole Cells by Asynchronous Localization of Photoswitching Emitters,” Biophys. J. 93 No. 11, 3285–3290 (2007).
[Crossref] [PubMed]

H. Bock, C. Geisler, C. A. Wurm, C. von Middendorff, S. Jakobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-Color Far-Field Fluorescence Nanoscopy Based on Photoswitchable Emitters,” Appl. Phys. B 88, 161–165 (2007).
[Crossref]

Keller, J.

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

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92, L67–L69 (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]

G. H. Patterson and J. Lippincott-Schwartz, “A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells,” Science 297, 1873–1877 (2002).
[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. Willing, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuose wave beams,” Nat. Methods 4,No. 11, 915–918 (2007).
[Crossref]

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schonle, and S. W. Hell, “Fluorescence Nanoscopy in Whole Cells by Asynchronous Localization of Photoswitching Emitters,” Biophys. J. 93 No. 11, 3285–3290 (2007).
[Crossref] [PubMed]

Middendorff, C. von

H. Bock, C. Geisler, C. A. Wurm, C. von Middendorff, S. Jakobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-Color Far-Field Fluorescence Nanoscopy Based on Photoswitchable Emitters,” Appl. Phys. B 88, 161–165 (2007).
[Crossref]

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schonle, and S. W. Hell, “Fluorescence Nanoscopy in Whole Cells by Asynchronous Localization of Photoswitching Emitters,” Biophys. J. 93 No. 11, 3285–3290 (2007).
[Crossref] [PubMed]

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]

G. H. Patterson and J. Lippincott-Schwartz, “A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells,” Science 297, 1873–1877 (2002).
[Crossref] [PubMed]

Rittweger, E.

S. E. Irvine, T. Staudt, E. Rittweger, J. Engelhardt, and S. W. Hell, “Direct Light-Driven Modulation of Luminescence from Mn-Doped ZnSe Quantum Dots,” Angew. Chem. Int. Ed. 47, Vol. 14, 2685–2688 (2008).
[Crossref]

Rizzoli, S. O.

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92, L67–L69 (2007).
[Crossref] [PubMed]

Rust, M. J.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3, No. 10, 793–796 (2006).
[Crossref] [PubMed]

Schonle, A.

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

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92, L67–L69 (2007).
[Crossref] [PubMed]

H. Bock, C. Geisler, C. A. Wurm, C. von Middendorff, S. Jakobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-Color Far-Field Fluorescence Nanoscopy Based on Photoswitchable Emitters,” Appl. Phys. B 88, 161–165 (2007).
[Crossref]

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schonle, and S. W. Hell, “Fluorescence Nanoscopy in Whole Cells by Asynchronous Localization of Photoswitching Emitters,” Biophys. J. 93 No. 11, 3285–3290 (2007).
[Crossref] [PubMed]

Sharonov, A.

A. Sharonov and R. M. Hochstrasser, “Wide-Field Subdiffraction Imaging by Accumulated Binding of Diffusing Probes,” Proc. Natl. Acad. Sci. 103, No. 50, 18911–18916 (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]

Staudt, T.

S. E. Irvine, T. Staudt, E. Rittweger, J. Engelhardt, and S. W. Hell, “Direct Light-Driven Modulation of Luminescence from Mn-Doped ZnSe Quantum Dots,” Angew. Chem. Int. Ed. 47, Vol. 14, 2685–2688 (2008).
[Crossref]

Stiel, A. C.

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schonle, and S. W. Hell, “Fluorescence Nanoscopy in Whole Cells by Asynchronous Localization of Photoswitching Emitters,” Biophys. J. 93 No. 11, 3285–3290 (2007).
[Crossref] [PubMed]

Ullal, C. K.

Wenzel, D.

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schonle, and S. W. Hell, “Fluorescence Nanoscopy in Whole Cells by Asynchronous Localization of Photoswitching Emitters,” Biophys. J. 93 No. 11, 3285–3290 (2007).
[Crossref] [PubMed]

Westphal, V.

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

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92, L67–L69 (2007).
[Crossref] [PubMed]

Wichmann, J.

Willing, K. I.

K. I. Willing, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuose wave beams,” Nat. Methods 4,No. 11, 915–918 (2007).
[Crossref]

Wurm, C. A.

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92, L67–L69 (2007).
[Crossref] [PubMed]

H. Bock, C. Geisler, C. A. Wurm, C. von Middendorff, S. Jakobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-Color Far-Field Fluorescence Nanoscopy Based on Photoswitchable Emitters,” Appl. Phys. B 88, 161–165 (2007).
[Crossref]

Zhuang, X.

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317, 1749–1753 (2007).
[Crossref] [PubMed]

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317, 1749–1753 (2007).
[Crossref] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3, No. 10, 793–796 (2006).
[Crossref] [PubMed]

M. Bates, T. R. Blosser, and X. Zhuang, “Short-Range Spectroscopic Ruler Based on a Single-Molecule Optical Switch,” Phys. Rev. Lett. 94, 108101(1–4) (2005).
[Crossref] [PubMed]

Angew. Chem. Int. Ed. (1)

S. E. Irvine, T. Staudt, E. Rittweger, J. Engelhardt, and S. W. Hell, “Direct Light-Driven Modulation of Luminescence from Mn-Doped ZnSe Quantum Dots,” Angew. Chem. Int. Ed. 47, Vol. 14, 2685–2688 (2008).
[Crossref]

Appl. Phys. B (1)

H. Bock, C. Geisler, C. A. Wurm, C. von Middendorff, S. Jakobs, A. Schonle, A. Egner, S. W. Hell, and C. Eggeling, “Two-Color Far-Field Fluorescence Nanoscopy Based on Photoswitchable Emitters,” Appl. Phys. B 88, 161–165 (2007).
[Crossref]

Biophys. J. (3)

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A. C. Stiel, S. Jakobs, C. Eggeling, A. Schonle, and S. W. Hell, “Fluorescence Nanoscopy in Whole Cells by Asynchronous Localization of Photoswitching Emitters,” Biophys. J. 93 No. 11, 3285–3290 (2007).
[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]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schonle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-Color Far-Field Fluorescence Nanoscopy,” Biophys. J. 92, L67–L69 (2007).
[Crossref] [PubMed]

Nat. Methods (2)

K. I. Willing, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuose wave beams,” Nat. Methods 4,No. 11, 915–918 (2007).
[Crossref]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3, No. 10, 793–796 (2006).
[Crossref] [PubMed]

Opt. Express (1)

Opt. Lett. (1)

Phys. Lett. A. (1)

S. W. Hell, “Strategy for far-field optical imaging and writing without diffraction limit,” Phys. Lett. A. 326, 140–145 (2004).
[Crossref]

Phys. Rev. Lett. (2)

M. Bates, T. R. Blosser, and X. Zhuang, “Short-Range Spectroscopic Ruler Based on a Single-Molecule Optical Switch,” Phys. Rev. Lett. 94, 108101(1–4) (2005).
[Crossref] [PubMed]

S. Bretschneider, C. Eggeling, and S. W. Hell, “Breaking the Diffraction Barrier in Fluorescence Microscopy by Optical Shelving,” Phys. Rev. Lett. 98, 218103(1–4) (2007).
[Crossref] [PubMed]

Proc. Natl. Acad. Sci. (1)

A. Sharonov and R. M. Hochstrasser, “Wide-Field Subdiffraction Imaging by Accumulated Binding of Diffusing Probes,” Proc. Natl. Acad. Sci. 103, No. 50, 18911–18916 (2006).
[Crossref] [PubMed]

Science (5)

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317, 1749–1753 (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]

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

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317, 1749–1753 (2007).
[Crossref] [PubMed]

G. H. Patterson and J. Lippincott-Schwartz, “A Photoactivatable GFP for Selective Photolabeling of Proteins and Cells,” Science 297, 1873–1877 (2002).
[Crossref] [PubMed]

Other (1)

R. W. Boyd, “Nonlinear Optics,” second edition, (Academic Press, 2003).

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Figures (3)

Fig. 1.
Fig. 1. Properties of a fluorophore exhibiting depletion upon saturation. a) Emission intensity (normalized to an undepleted fluorophore) as a function of excitation intensity for several values of α. b) The maximal emission yield (relative to an undepleted fluorophore) as a function of α.
Fig. 2.
Fig. 2. Excitation and (dashed-dot blue line) and emission profiles (dashed black line) along the X axis for a fluorophore exhibiting emission depletion upon saturation. The overall detected intensity through a confocal pinhole as a function of its position along the X axis (solid red line), exhibiting deep dips between the central lobe and the sidelobes, shows clearly that at x=0 efficient rejection of the sidelobe emission is possible. The inset shows a blow up of the origin to emphasis that the illumination pattern (blue/dash-dot) is non-vanishing at the center. The parameters used for this plot are (Id /Imax = 0.04, Γ/∆ = 3 and α = 4)
Fig. 3.
Fig. 3. a) The width of the central lobe and the light contrast (ratio of signal in the central lobe to that in the outer ring) as a function of the excitation intensity. b) the distance from the central lobe to the peak emission in the outer ring as a function of excitation intensity.

Equations (8)

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

F(I)=(1eIIem)·eIId.
Fm(α)=α1+α·11+α(1α).
I(r)=Imax·e4·(8r2Γ2e2r2Γ2+δe2r2Γ2),
δ=4e IemImax ln (1+α).
MXwΔΓΔ Id2e.Imax 1+ln((1+α)α).,
dNRdt=NRτradNRNDτdep+αI(1NR)
dNDdt=NDτrel+βI(1ND)
NR=τradαI1+τrelβIτrad(1+τrelβI)τdep+τradαI

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