Abstract

We combine far-field fluorescence nanoscopy through serialized recording of switchable emitters with polarization-sensitive fluorescence detection. In addition to imaging with nanoscale spatial resolution, this technique allows determination of the fluorescence anisotropy of each detected dipole emitter and thus an estimate of its rotational mobility. Sub-populations of fluorescent markers can thus be separated based on their interaction with the sample. We applied this new functional nanoscopy to imaging of living mammalian cells.

© 2008 Optical Society of America

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  1. E. Abbe, "Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung," Arch. Mikr. Anat. 9, 413-468 (1873).
    [CrossRef]
  2. S. W. Hell, "Improvement of lateral resolution in far-field light microscopy using two-photon excitation with offset beams," Opt. Commun. 106, 19-24 (1994).
    [CrossRef]
  3. 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]
  4. S. W. Hell, "Toward fluorescence nanoscopy," Nature Biotechnol. 21, 1347-1355 (2003).
    [CrossRef]
  5. S. W. Hell, "Far-Field Optical Nanoscopy," Science 316, 1153-1158 (2007).
    [CrossRef] [PubMed]
  6. 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]
  7. 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]
  8. M. J. Rust, M. Bates, and X. Zhuang, "Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)," Nat. Methods 3, 793-796 (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. A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A.-C. Stiel, S. Jakobs, C. Eggeling, A. Schönle, and S. W. Hell, "Fluorescence nanoscopy in whole cells by asnychronous localization of photoswitching emitters," Biophys. J. 93, 3285-3290 (2007).
    [CrossRef] [PubMed]
  11. W. Heisenberg, The Physical Principles of the Quantum Theory (Chicago Univ Press, Chicago, 1930).
  12. N. Bobroff, "Position measurement with a resolution and noise-limited instrument," Rev. Sci. Instrum. 57, 1152-1157 (1986).
    [CrossRef]
  13. E. Betzig, "Proposed method for molecular optical imaging," Opt. Lett. 20, 237-239 (1995).
    [CrossRef] [PubMed]
  14. S. W. Hell, J. Soukka, and P. E. Hänninen, "Two- and multiphoton detection as an imaging mode and means of increasing the resolution in far-field light microscopy," Bioimaging 3, 65-69 (1995).
    [CrossRef]
  15. T. Schmidt, G. J. Schutz, W. Baumgartner, H. J. Gruber, and H. Schindler, "Imaging of single molecule diffusion," Proc. Natl. Acad. Sci. USA 93, 2926-9 (1996).
    [CrossRef] [PubMed]
  16. T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, "Ultrahigh-resolution multicolor colocalization of single fluorescent probes," Proc. Natl. Acad. Sci. USA 97, 9461-9466 (2000).
    [CrossRef] [PubMed]
  17. R. E. Thompson, D. R. Larson, and W. W. Webb, "Precise nanometer localization analysis for individual fluorescent probes," Biophys. J. 82, 2775-2783 (2002).
    [CrossRef] [PubMed]
  18. W. E. Moerner and L. Kador, "Optical detection and spectroscopy of single molecules in a solid," Phys. Rev. Lett. 62, 2535-2538 (1989).
    [CrossRef] [PubMed]
  19. M. Orrit and J. Bernard, "Single pentacene molecules detected by fluorescence excitation in a p-terphenyl crystal," Phys. Rev. Lett. 65, 2716-2719 (1990).
    [CrossRef] [PubMed]
  20. E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper, "Detection of single fluorescent molecules," Chem. Phys. Lett. 174, 553-557 (1990).
    [CrossRef]
  21. T. Ha, T. Enderle, D. F. Ogletree, D. S. Chemla, P. R. Selvin, and S. Weiss, "Probing the interaction between two single molecules: Fluorescence resonance energy transfer between a single donor and a single acceptor," Proc. Natl. Acad. Sci. USA 93, 6264-6268 (1996).
    [CrossRef] [PubMed]
  22. A. Schönle and S. W. Hell "Fluorescence nanoscopy goes multicolor," Nature Biotechnol. 25, 1234-1235 (2007).
    [CrossRef]
  23. M. Bossi, J. Fölling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schönle, and S. W. Hell, "Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species," Nano Lett. 8, 2463-2468 (2008).
    [CrossRef] [PubMed]
  24. M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S. W. Hell, and S. Jakobs, "Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy," Nat. Biotechnol. 26, 1035 - 1040 (2008).
    [CrossRef] [PubMed]
  25. T. Ha, T. Enderle, D. S. Chemla, P. R. Selvin, and S. Weiss, "Single molecule dynamics studied by polarization modulation," Phys. Rev. Lett. 77, 3979-3982 (1996).
    [CrossRef] [PubMed]
  26. J. Schaffer, A. Volkmer, C. Eggeling, V. Subramaniam, G. Striker, and C. A. M. Seidel, "Identification of single molecules in aqueous solution by time-resolved fluorescence anisotropy," J. Phys. Chem. A 103, 331-336 (1999).
    [CrossRef]
  27. J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, "Photochromic Rhodamines Provide Nanoscopy with Optical Sectioning," Angew. Chem. Int. Ed. 46, 6266-6270 (2007).
    [CrossRef]
  28. A. van Blaaderen and A. Vrij, "Synthesis and Characterization of Colloidal Dispersions of Fluorescent, Monodisperse Silica Spheres," Langmuir 8, 2921-2931 (1992).
    [CrossRef]
  29. J. Fölling, V. Belov, D. Riedel, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell "Fluorescence Nanoscopy with Optical Sectioning by Two-Photon Induced Molecular Switching using Continuous-Wave Lasers," ChemPhysChem 9, 321 - 326 (2008).
    [CrossRef] [PubMed]
  30. A. C. Stiel, M. Andresen, H. Bock, M. Hilbert, J. Schilde, A. Schönle, C. Eggeling, A. Egner, S. W. Hell, and S. Jakobs, "Generation of Monomeric Reversibly Switchable Red Fluorescent Proteins for Far-Field Fluorescence Nanoscopy," Biophys. J. 95, 2989-2997 (2008).
    [CrossRef] [PubMed]
  31. J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. Röcker, A. Salih, K.-D. Spindler, and G. U. Nienhaus, "EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion," Proc.Natl.Acad.Sci.USA 101, 15905-15910 (2004).
    [CrossRef] [PubMed]
  32. P. Lamesch, N. Li, S. Milstein, C. Fan, T. Hao, G. Szabo, Z. Hu, K. Venkatesan, G. Bethel, P. Martin, J. Rogers, S. Lawlor, S. McLaren, A. Dricot, H. Borick, M. E. Cusick, J. Vandenhaute, I. Dunham, D. E. Hill, and M. Vidal, "hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes," Genomics 89, 307-315 (2007).
    [CrossRef] [PubMed]
  33. C. Geisler, A. Schönle, C. von Middendorff, H. Bock, C. Eggeling, A. Egner, and S. W. Hell "Resolution of λ/10 in fluorescence microscopy using fast single molecule photo-switching," Appl. Phys. A 88, 223-226 (2007).
    [CrossRef]
  34. J. Enderlein, "Theoretical study of detection of a dipole emitter through an objectiv with high numerical aperture," Opt. Lett. 25, 634-636 (2000).
    [CrossRef]
  35. K. Bacia and P. Schwille, "A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy," Methods 29, 74-85 (2003).
    [CrossRef] [PubMed]
  36. T. L. Hill and M. W. Kischner, "Subunit treadmilling of microtubules or actin in the presence of cellular barriers: possible conversion of chemical free energy into mechanical work," Proc. Natl. Acad. Sci. USA 79, 490-494 (1982).
    [CrossRef] [PubMed]
  37. C. Zander, M. Sauer, K. H. Drexhage, D.-S. Ko, A. Schulz, J. Wolfrum, L. Brand, C. Eggeling, and C. A. M. Seidel, "Detection and characterization of single molecules in aqueous solution," Appl. Phys. B 63, 517-523 (1996).
  38. J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell "Fluorescence nanoscopy by ground-state depletion and single-molecule return," Nat. Meth. 5, 943 - 945 (2008).
    [CrossRef]

2008

M. Bossi, J. Fölling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schönle, and S. W. Hell, "Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species," Nano Lett. 8, 2463-2468 (2008).
[CrossRef] [PubMed]

M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S. W. Hell, and S. Jakobs, "Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy," Nat. Biotechnol. 26, 1035 - 1040 (2008).
[CrossRef] [PubMed]

J. Fölling, V. Belov, D. Riedel, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell "Fluorescence Nanoscopy with Optical Sectioning by Two-Photon Induced Molecular Switching using Continuous-Wave Lasers," ChemPhysChem 9, 321 - 326 (2008).
[CrossRef] [PubMed]

A. C. Stiel, M. Andresen, H. Bock, M. Hilbert, J. Schilde, A. Schönle, C. Eggeling, A. Egner, S. W. Hell, and S. Jakobs, "Generation of Monomeric Reversibly Switchable Red Fluorescent Proteins for Far-Field Fluorescence Nanoscopy," Biophys. J. 95, 2989-2997 (2008).
[CrossRef] [PubMed]

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell "Fluorescence nanoscopy by ground-state depletion and single-molecule return," Nat. Meth. 5, 943 - 945 (2008).
[CrossRef]

2007

P. Lamesch, N. Li, S. Milstein, C. Fan, T. Hao, G. Szabo, Z. Hu, K. Venkatesan, G. Bethel, P. Martin, J. Rogers, S. Lawlor, S. McLaren, A. Dricot, H. Borick, M. E. Cusick, J. Vandenhaute, I. Dunham, D. E. Hill, and M. Vidal, "hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes," Genomics 89, 307-315 (2007).
[CrossRef] [PubMed]

C. Geisler, A. Schönle, C. von Middendorff, H. Bock, C. Eggeling, A. Egner, and S. W. Hell "Resolution of λ/10 in fluorescence microscopy using fast single molecule photo-switching," Appl. Phys. A 88, 223-226 (2007).
[CrossRef]

S. W. Hell, "Far-Field Optical Nanoscopy," Science 316, 1153-1158 (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. Schönle, and S. W. Hell, "Fluorescence nanoscopy in whole cells by asnychronous localization of photoswitching emitters," Biophys. J. 93, 3285-3290 (2007).
[CrossRef] [PubMed]

J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, "Photochromic Rhodamines Provide Nanoscopy with Optical Sectioning," Angew. Chem. Int. Ed. 46, 6266-6270 (2007).
[CrossRef]

A. Schönle and S. W. Hell "Fluorescence nanoscopy goes multicolor," Nature Biotechnol. 25, 1234-1235 (2007).
[CrossRef]

2006

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. Methods 3, 793-796 (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]

2004

J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. Röcker, A. Salih, K.-D. Spindler, and G. U. Nienhaus, "EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion," Proc.Natl.Acad.Sci.USA 101, 15905-15910 (2004).
[CrossRef] [PubMed]

2003

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

K. Bacia and P. Schwille, "A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy," Methods 29, 74-85 (2003).
[CrossRef] [PubMed]

2002

R. E. Thompson, D. R. Larson, and W. W. Webb, "Precise nanometer localization analysis for individual fluorescent probes," Biophys. J. 82, 2775-2783 (2002).
[CrossRef] [PubMed]

2000

T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, "Ultrahigh-resolution multicolor colocalization of single fluorescent probes," Proc. Natl. Acad. Sci. USA 97, 9461-9466 (2000).
[CrossRef] [PubMed]

J. Enderlein, "Theoretical study of detection of a dipole emitter through an objectiv with high numerical aperture," Opt. Lett. 25, 634-636 (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]

1999

J. Schaffer, A. Volkmer, C. Eggeling, V. Subramaniam, G. Striker, and C. A. M. Seidel, "Identification of single molecules in aqueous solution by time-resolved fluorescence anisotropy," J. Phys. Chem. A 103, 331-336 (1999).
[CrossRef]

1996

T. Ha, T. Enderle, D. S. Chemla, P. R. Selvin, and S. Weiss, "Single molecule dynamics studied by polarization modulation," Phys. Rev. Lett. 77, 3979-3982 (1996).
[CrossRef] [PubMed]

T. Ha, T. Enderle, D. F. Ogletree, D. S. Chemla, P. R. Selvin, and S. Weiss, "Probing the interaction between two single molecules: Fluorescence resonance energy transfer between a single donor and a single acceptor," Proc. Natl. Acad. Sci. USA 93, 6264-6268 (1996).
[CrossRef] [PubMed]

T. Schmidt, G. J. Schutz, W. Baumgartner, H. J. Gruber, and H. Schindler, "Imaging of single molecule diffusion," Proc. Natl. Acad. Sci. USA 93, 2926-9 (1996).
[CrossRef] [PubMed]

C. Zander, M. Sauer, K. H. Drexhage, D.-S. Ko, A. Schulz, J. Wolfrum, L. Brand, C. Eggeling, and C. A. M. Seidel, "Detection and characterization of single molecules in aqueous solution," Appl. Phys. B 63, 517-523 (1996).

1995

S. W. Hell, J. Soukka, and P. E. Hänninen, "Two- and multiphoton detection as an imaging mode and means of increasing the resolution in far-field light microscopy," Bioimaging 3, 65-69 (1995).
[CrossRef]

E. Betzig, "Proposed method for molecular optical imaging," Opt. Lett. 20, 237-239 (1995).
[CrossRef] [PubMed]

1994

S. W. Hell, "Improvement of lateral resolution in far-field light microscopy using two-photon excitation with offset beams," Opt. Commun. 106, 19-24 (1994).
[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]

1992

A. van Blaaderen and A. Vrij, "Synthesis and Characterization of Colloidal Dispersions of Fluorescent, Monodisperse Silica Spheres," Langmuir 8, 2921-2931 (1992).
[CrossRef]

1990

M. Orrit and J. Bernard, "Single pentacene molecules detected by fluorescence excitation in a p-terphenyl crystal," Phys. Rev. Lett. 65, 2716-2719 (1990).
[CrossRef] [PubMed]

E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper, "Detection of single fluorescent molecules," Chem. Phys. Lett. 174, 553-557 (1990).
[CrossRef]

1989

W. E. Moerner and L. Kador, "Optical detection and spectroscopy of single molecules in a solid," Phys. Rev. Lett. 62, 2535-2538 (1989).
[CrossRef] [PubMed]

1986

N. Bobroff, "Position measurement with a resolution and noise-limited instrument," Rev. Sci. Instrum. 57, 1152-1157 (1986).
[CrossRef]

1982

T. L. Hill and M. W. Kischner, "Subunit treadmilling of microtubules or actin in the presence of cellular barriers: possible conversion of chemical free energy into mechanical work," Proc. Natl. Acad. Sci. USA 79, 490-494 (1982).
[CrossRef] [PubMed]

1873

E. Abbe, "Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung," Arch. Mikr. Anat. 9, 413-468 (1873).
[CrossRef]

Abbe, E.

E. Abbe, "Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung," Arch. Mikr. Anat. 9, 413-468 (1873).
[CrossRef]

Alivisatos, A. P.

T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, "Ultrahigh-resolution multicolor colocalization of single fluorescent probes," Proc. Natl. Acad. Sci. USA 97, 9461-9466 (2000).
[CrossRef] [PubMed]

Andresen, M.

M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S. W. Hell, and S. Jakobs, "Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy," Nat. Biotechnol. 26, 1035 - 1040 (2008).
[CrossRef] [PubMed]

A. C. Stiel, M. Andresen, H. Bock, M. Hilbert, J. Schilde, A. Schönle, C. Eggeling, A. Egner, S. W. Hell, and S. Jakobs, "Generation of Monomeric Reversibly Switchable Red Fluorescent Proteins for Far-Field Fluorescence Nanoscopy," Biophys. J. 95, 2989-2997 (2008).
[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. Schönle, and S. W. Hell, "Fluorescence nanoscopy in whole cells by asnychronous localization of photoswitching emitters," Biophys. J. 93, 3285-3290 (2007).
[CrossRef] [PubMed]

Bacia, K.

K. Bacia and P. Schwille, "A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy," Methods 29, 74-85 (2003).
[CrossRef] [PubMed]

Bates, M.

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

Baumgartner, W.

T. Schmidt, G. J. Schutz, W. Baumgartner, H. J. Gruber, and H. Schindler, "Imaging of single molecule diffusion," Proc. Natl. Acad. Sci. USA 93, 2926-9 (1996).
[CrossRef] [PubMed]

Belov, V.

J. Fölling, V. Belov, D. Riedel, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell "Fluorescence Nanoscopy with Optical Sectioning by Two-Photon Induced Molecular Switching using Continuous-Wave Lasers," ChemPhysChem 9, 321 - 326 (2008).
[CrossRef] [PubMed]

J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, "Photochromic Rhodamines Provide Nanoscopy with Optical Sectioning," Angew. Chem. Int. Ed. 46, 6266-6270 (2007).
[CrossRef]

Belov, V. N.

M. Bossi, J. Fölling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schönle, and S. W. Hell, "Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species," Nano Lett. 8, 2463-2468 (2008).
[CrossRef] [PubMed]

Bernard, J.

M. Orrit and J. Bernard, "Single pentacene molecules detected by fluorescence excitation in a p-terphenyl crystal," Phys. Rev. Lett. 65, 2716-2719 (1990).
[CrossRef] [PubMed]

Bethel, G.

P. Lamesch, N. Li, S. Milstein, C. Fan, T. Hao, G. Szabo, Z. Hu, K. Venkatesan, G. Bethel, P. Martin, J. Rogers, S. Lawlor, S. McLaren, A. Dricot, H. Borick, M. E. Cusick, J. Vandenhaute, I. Dunham, D. E. Hill, and M. Vidal, "hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes," Genomics 89, 307-315 (2007).
[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]

E. Betzig, "Proposed method for molecular optical imaging," Opt. Lett. 20, 237-239 (1995).
[CrossRef] [PubMed]

Bobroff, N.

N. Bobroff, "Position measurement with a resolution and noise-limited instrument," Rev. Sci. Instrum. 57, 1152-1157 (1986).
[CrossRef]

Bock, H.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell "Fluorescence nanoscopy by ground-state depletion and single-molecule return," Nat. Meth. 5, 943 - 945 (2008).
[CrossRef]

A. C. Stiel, M. Andresen, H. Bock, M. Hilbert, J. Schilde, A. Schönle, C. Eggeling, A. Egner, S. W. Hell, and S. Jakobs, "Generation of Monomeric Reversibly Switchable Red Fluorescent Proteins for Far-Field Fluorescence Nanoscopy," Biophys. J. 95, 2989-2997 (2008).
[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. Schönle, and S. W. Hell, "Fluorescence nanoscopy in whole cells by asnychronous localization of photoswitching emitters," Biophys. J. 93, 3285-3290 (2007).
[CrossRef] [PubMed]

C. Geisler, A. Schönle, C. von Middendorff, H. Bock, C. Eggeling, A. Egner, and S. W. Hell "Resolution of λ/10 in fluorescence microscopy using fast single molecule photo-switching," Appl. Phys. A 88, 223-226 (2007).
[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]

Borick, H.

P. Lamesch, N. Li, S. Milstein, C. Fan, T. Hao, G. Szabo, Z. Hu, K. Venkatesan, G. Bethel, P. Martin, J. Rogers, S. Lawlor, S. McLaren, A. Dricot, H. Borick, M. E. Cusick, J. Vandenhaute, I. Dunham, D. E. Hill, and M. Vidal, "hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes," Genomics 89, 307-315 (2007).
[CrossRef] [PubMed]

Bossi, M.

M. Bossi, J. Fölling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schönle, and S. W. Hell, "Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species," Nano Lett. 8, 2463-2468 (2008).
[CrossRef] [PubMed]

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell "Fluorescence nanoscopy by ground-state depletion and single-molecule return," Nat. Meth. 5, 943 - 945 (2008).
[CrossRef]

J. Fölling, V. Belov, D. Riedel, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell "Fluorescence Nanoscopy with Optical Sectioning by Two-Photon Induced Molecular Switching using Continuous-Wave Lasers," ChemPhysChem 9, 321 - 326 (2008).
[CrossRef] [PubMed]

J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, "Photochromic Rhodamines Provide Nanoscopy with Optical Sectioning," Angew. Chem. Int. Ed. 46, 6266-6270 (2007).
[CrossRef]

Boyarskiy, V. P.

M. Bossi, J. Fölling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schönle, and S. W. Hell, "Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species," Nano Lett. 8, 2463-2468 (2008).
[CrossRef] [PubMed]

Brand, L.

C. Zander, M. Sauer, K. H. Drexhage, D.-S. Ko, A. Schulz, J. Wolfrum, L. Brand, C. Eggeling, and C. A. M. Seidel, "Detection and characterization of single molecules in aqueous solution," Appl. Phys. B 63, 517-523 (1996).

Chemla, D. S.

T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, "Ultrahigh-resolution multicolor colocalization of single fluorescent probes," Proc. Natl. Acad. Sci. USA 97, 9461-9466 (2000).
[CrossRef] [PubMed]

T. Ha, T. Enderle, D. S. Chemla, P. R. Selvin, and S. Weiss, "Single molecule dynamics studied by polarization modulation," Phys. Rev. Lett. 77, 3979-3982 (1996).
[CrossRef] [PubMed]

T. Ha, T. Enderle, D. F. Ogletree, D. S. Chemla, P. R. Selvin, and S. Weiss, "Probing the interaction between two single molecules: Fluorescence resonance energy transfer between a single donor and a single acceptor," Proc. Natl. Acad. Sci. USA 93, 6264-6268 (1996).
[CrossRef] [PubMed]

Cusick, M. E.

P. Lamesch, N. Li, S. Milstein, C. Fan, T. Hao, G. Szabo, Z. Hu, K. Venkatesan, G. Bethel, P. Martin, J. Rogers, S. Lawlor, S. McLaren, A. Dricot, H. Borick, M. E. Cusick, J. Vandenhaute, I. Dunham, D. E. Hill, and M. Vidal, "hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes," Genomics 89, 307-315 (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]

Davis, L. M.

E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper, "Detection of single fluorescent molecules," Chem. Phys. Lett. 174, 553-557 (1990).
[CrossRef]

Drexhage, K. H.

C. Zander, M. Sauer, K. H. Drexhage, D.-S. Ko, A. Schulz, J. Wolfrum, L. Brand, C. Eggeling, and C. A. M. Seidel, "Detection and characterization of single molecules in aqueous solution," Appl. Phys. B 63, 517-523 (1996).

Dricot, A.

P. Lamesch, N. Li, S. Milstein, C. Fan, T. Hao, G. Szabo, Z. Hu, K. Venkatesan, G. Bethel, P. Martin, J. Rogers, S. Lawlor, S. McLaren, A. Dricot, H. Borick, M. E. Cusick, J. Vandenhaute, I. Dunham, D. E. Hill, and M. Vidal, "hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes," Genomics 89, 307-315 (2007).
[CrossRef] [PubMed]

Dunham, I.

P. Lamesch, N. Li, S. Milstein, C. Fan, T. Hao, G. Szabo, Z. Hu, K. Venkatesan, G. Bethel, P. Martin, J. Rogers, S. Lawlor, S. McLaren, A. Dricot, H. Borick, M. E. Cusick, J. Vandenhaute, I. Dunham, D. E. Hill, and M. Vidal, "hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes," Genomics 89, 307-315 (2007).
[CrossRef] [PubMed]

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.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell "Fluorescence nanoscopy by ground-state depletion and single-molecule return," Nat. Meth. 5, 943 - 945 (2008).
[CrossRef]

M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S. W. Hell, and S. Jakobs, "Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy," Nat. Biotechnol. 26, 1035 - 1040 (2008).
[CrossRef] [PubMed]

M. Bossi, J. Fölling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schönle, and S. W. Hell, "Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species," Nano Lett. 8, 2463-2468 (2008).
[CrossRef] [PubMed]

A. C. Stiel, M. Andresen, H. Bock, M. Hilbert, J. Schilde, A. Schönle, C. Eggeling, A. Egner, S. W. Hell, and S. Jakobs, "Generation of Monomeric Reversibly Switchable Red Fluorescent Proteins for Far-Field Fluorescence Nanoscopy," Biophys. J. 95, 2989-2997 (2008).
[CrossRef] [PubMed]

J. Fölling, V. Belov, D. Riedel, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell "Fluorescence Nanoscopy with Optical Sectioning by Two-Photon Induced Molecular Switching using Continuous-Wave Lasers," ChemPhysChem 9, 321 - 326 (2008).
[CrossRef] [PubMed]

C. Geisler, A. Schönle, C. von Middendorff, H. Bock, C. Eggeling, A. Egner, and S. W. Hell "Resolution of λ/10 in fluorescence microscopy using fast single molecule photo-switching," Appl. Phys. A 88, 223-226 (2007).
[CrossRef]

J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, "Photochromic Rhodamines Provide Nanoscopy with Optical Sectioning," Angew. Chem. Int. Ed. 46, 6266-6270 (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. Schönle, and S. W. Hell, "Fluorescence nanoscopy in whole cells by asnychronous localization of photoswitching emitters," Biophys. J. 93, 3285-3290 (2007).
[CrossRef] [PubMed]

J. Schaffer, A. Volkmer, C. Eggeling, V. Subramaniam, G. Striker, and C. A. M. Seidel, "Identification of single molecules in aqueous solution by time-resolved fluorescence anisotropy," J. Phys. Chem. A 103, 331-336 (1999).
[CrossRef]

C. Zander, M. Sauer, K. H. Drexhage, D.-S. Ko, A. Schulz, J. Wolfrum, L. Brand, C. Eggeling, and C. A. M. Seidel, "Detection and characterization of single molecules in aqueous solution," Appl. Phys. B 63, 517-523 (1996).

Egner, A.

J. Fölling, V. Belov, D. Riedel, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell "Fluorescence Nanoscopy with Optical Sectioning by Two-Photon Induced Molecular Switching using Continuous-Wave Lasers," ChemPhysChem 9, 321 - 326 (2008).
[CrossRef] [PubMed]

M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S. W. Hell, and S. Jakobs, "Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy," Nat. Biotechnol. 26, 1035 - 1040 (2008).
[CrossRef] [PubMed]

A. C. Stiel, M. Andresen, H. Bock, M. Hilbert, J. Schilde, A. Schönle, C. Eggeling, A. Egner, S. W. Hell, and S. Jakobs, "Generation of Monomeric Reversibly Switchable Red Fluorescent Proteins for Far-Field Fluorescence Nanoscopy," Biophys. J. 95, 2989-2997 (2008).
[CrossRef] [PubMed]

M. Bossi, J. Fölling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schönle, and S. W. Hell, "Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species," Nano Lett. 8, 2463-2468 (2008).
[CrossRef] [PubMed]

J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, "Photochromic Rhodamines Provide Nanoscopy with Optical Sectioning," Angew. Chem. Int. Ed. 46, 6266-6270 (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. Schönle, and S. W. Hell, "Fluorescence nanoscopy in whole cells by asnychronous localization of photoswitching emitters," Biophys. J. 93, 3285-3290 (2007).
[CrossRef] [PubMed]

C. Geisler, A. Schönle, C. von Middendorff, H. Bock, C. Eggeling, A. Egner, and S. W. Hell "Resolution of λ/10 in fluorescence microscopy using fast single molecule photo-switching," Appl. Phys. A 88, 223-226 (2007).
[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]

Enderle, T.

T. Ha, T. Enderle, D. F. Ogletree, D. S. Chemla, P. R. Selvin, and S. Weiss, "Probing the interaction between two single molecules: Fluorescence resonance energy transfer between a single donor and a single acceptor," Proc. Natl. Acad. Sci. USA 93, 6264-6268 (1996).
[CrossRef] [PubMed]

T. Ha, T. Enderle, D. S. Chemla, P. R. Selvin, and S. Weiss, "Single molecule dynamics studied by polarization modulation," Phys. Rev. Lett. 77, 3979-3982 (1996).
[CrossRef] [PubMed]

Enderlein, J.

Fan, C.

P. Lamesch, N. Li, S. Milstein, C. Fan, T. Hao, G. Szabo, Z. Hu, K. Venkatesan, G. Bethel, P. Martin, J. Rogers, S. Lawlor, S. McLaren, A. Dricot, H. Borick, M. E. Cusick, J. Vandenhaute, I. Dunham, D. E. Hill, and M. Vidal, "hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes," Genomics 89, 307-315 (2007).
[CrossRef] [PubMed]

Fölling, J.

M. Bossi, J. Fölling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schönle, and S. W. Hell, "Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species," Nano Lett. 8, 2463-2468 (2008).
[CrossRef] [PubMed]

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell "Fluorescence nanoscopy by ground-state depletion and single-molecule return," Nat. Meth. 5, 943 - 945 (2008).
[CrossRef]

M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S. W. Hell, and S. Jakobs, "Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy," Nat. Biotechnol. 26, 1035 - 1040 (2008).
[CrossRef] [PubMed]

J. Fölling, V. Belov, D. Riedel, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell "Fluorescence Nanoscopy with Optical Sectioning by Two-Photon Induced Molecular Switching using Continuous-Wave Lasers," ChemPhysChem 9, 321 - 326 (2008).
[CrossRef] [PubMed]

J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, "Photochromic Rhodamines Provide Nanoscopy with Optical Sectioning," Angew. Chem. Int. Ed. 46, 6266-6270 (2007).
[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. Schönle, and S. W. Hell, "Fluorescence nanoscopy in whole cells by asnychronous localization of photoswitching emitters," Biophys. J. 93, 3285-3290 (2007).
[CrossRef] [PubMed]

C. Geisler, A. Schönle, C. von Middendorff, H. Bock, C. Eggeling, A. Egner, and S. W. Hell "Resolution of λ/10 in fluorescence microscopy using fast single molecule photo-switching," Appl. Phys. A 88, 223-226 (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]

Gruber, H. J.

T. Schmidt, G. J. Schutz, W. Baumgartner, H. J. Gruber, and H. Schindler, "Imaging of single molecule diffusion," Proc. Natl. Acad. Sci. USA 93, 2926-9 (1996).
[CrossRef] [PubMed]

Ha, T.

T. Ha, T. Enderle, D. F. Ogletree, D. S. Chemla, P. R. Selvin, and S. Weiss, "Probing the interaction between two single molecules: Fluorescence resonance energy transfer between a single donor and a single acceptor," Proc. Natl. Acad. Sci. USA 93, 6264-6268 (1996).
[CrossRef] [PubMed]

T. Ha, T. Enderle, D. S. Chemla, P. R. Selvin, and S. Weiss, "Single molecule dynamics studied by polarization modulation," Phys. Rev. Lett. 77, 3979-3982 (1996).
[CrossRef] [PubMed]

Hänninen, P. E.

S. W. Hell, J. Soukka, and P. E. Hänninen, "Two- and multiphoton detection as an imaging mode and means of increasing the resolution in far-field light microscopy," Bioimaging 3, 65-69 (1995).
[CrossRef]

Hao, T.

P. Lamesch, N. Li, S. Milstein, C. Fan, T. Hao, G. Szabo, Z. Hu, K. Venkatesan, G. Bethel, P. Martin, J. Rogers, S. Lawlor, S. McLaren, A. Dricot, H. Borick, M. E. Cusick, J. Vandenhaute, I. Dunham, D. E. Hill, and M. Vidal, "hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes," Genomics 89, 307-315 (2007).
[CrossRef] [PubMed]

Hein, B.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell "Fluorescence nanoscopy by ground-state depletion and single-molecule return," Nat. Meth. 5, 943 - 945 (2008).
[CrossRef]

Hell, S. W.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell "Fluorescence nanoscopy by ground-state depletion and single-molecule return," Nat. Meth. 5, 943 - 945 (2008).
[CrossRef]

M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S. W. Hell, and S. Jakobs, "Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy," Nat. Biotechnol. 26, 1035 - 1040 (2008).
[CrossRef] [PubMed]

A. C. Stiel, M. Andresen, H. Bock, M. Hilbert, J. Schilde, A. Schönle, C. Eggeling, A. Egner, S. W. Hell, and S. Jakobs, "Generation of Monomeric Reversibly Switchable Red Fluorescent Proteins for Far-Field Fluorescence Nanoscopy," Biophys. J. 95, 2989-2997 (2008).
[CrossRef] [PubMed]

M. Bossi, J. Fölling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schönle, and S. W. Hell, "Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species," Nano Lett. 8, 2463-2468 (2008).
[CrossRef] [PubMed]

J. Fölling, V. Belov, D. Riedel, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell "Fluorescence Nanoscopy with Optical Sectioning by Two-Photon Induced Molecular Switching using Continuous-Wave Lasers," ChemPhysChem 9, 321 - 326 (2008).
[CrossRef] [PubMed]

A. Schönle and S. W. Hell "Fluorescence nanoscopy goes multicolor," Nature Biotechnol. 25, 1234-1235 (2007).
[CrossRef]

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

C. Geisler, A. Schönle, C. von Middendorff, H. Bock, C. Eggeling, A. Egner, and S. W. Hell "Resolution of λ/10 in fluorescence microscopy using fast single molecule photo-switching," Appl. Phys. A 88, 223-226 (2007).
[CrossRef]

J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, "Photochromic Rhodamines Provide Nanoscopy with Optical Sectioning," Angew. Chem. Int. Ed. 46, 6266-6270 (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. Schönle, and S. W. Hell, "Fluorescence nanoscopy in whole cells by asnychronous localization of photoswitching emitters," Biophys. J. 93, 3285-3290 (2007).
[CrossRef] [PubMed]

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

S. W. Hell, J. Soukka, and P. E. Hänninen, "Two- and multiphoton detection as an imaging mode and means of increasing the resolution in far-field light microscopy," Bioimaging 3, 65-69 (1995).
[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]

S. W. Hell, "Improvement of lateral resolution in far-field light microscopy using two-photon excitation with offset beams," Opt. Commun. 106, 19-24 (1994).
[CrossRef]

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]

Hilbert, M.

A. C. Stiel, M. Andresen, H. Bock, M. Hilbert, J. Schilde, A. Schönle, C. Eggeling, A. Egner, S. W. Hell, and S. Jakobs, "Generation of Monomeric Reversibly Switchable Red Fluorescent Proteins for Far-Field Fluorescence Nanoscopy," Biophys. J. 95, 2989-2997 (2008).
[CrossRef] [PubMed]

Hill, D. E.

P. Lamesch, N. Li, S. Milstein, C. Fan, T. Hao, G. Szabo, Z. Hu, K. Venkatesan, G. Bethel, P. Martin, J. Rogers, S. Lawlor, S. McLaren, A. Dricot, H. Borick, M. E. Cusick, J. Vandenhaute, I. Dunham, D. E. Hill, and M. Vidal, "hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes," Genomics 89, 307-315 (2007).
[CrossRef] [PubMed]

Hill, T. L.

T. L. Hill and M. W. Kischner, "Subunit treadmilling of microtubules or actin in the presence of cellular barriers: possible conversion of chemical free energy into mechanical work," Proc. Natl. Acad. Sci. USA 79, 490-494 (1982).
[CrossRef] [PubMed]

Hu, Z.

P. Lamesch, N. Li, S. Milstein, C. Fan, T. Hao, G. Szabo, Z. Hu, K. Venkatesan, G. Bethel, P. Martin, J. Rogers, S. Lawlor, S. McLaren, A. Dricot, H. Borick, M. E. Cusick, J. Vandenhaute, I. Dunham, D. E. Hill, and M. Vidal, "hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes," Genomics 89, 307-315 (2007).
[CrossRef] [PubMed]

Ivanchenko, S.

J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. Röcker, A. Salih, K.-D. Spindler, and G. U. Nienhaus, "EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion," Proc.Natl.Acad.Sci.USA 101, 15905-15910 (2004).
[CrossRef] [PubMed]

Jakobs, S.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell "Fluorescence nanoscopy by ground-state depletion and single-molecule return," Nat. Meth. 5, 943 - 945 (2008).
[CrossRef]

M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S. W. Hell, and S. Jakobs, "Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy," Nat. Biotechnol. 26, 1035 - 1040 (2008).
[CrossRef] [PubMed]

A. C. Stiel, M. Andresen, H. Bock, M. Hilbert, J. Schilde, A. Schönle, C. Eggeling, A. Egner, S. W. Hell, and S. Jakobs, "Generation of Monomeric Reversibly Switchable Red Fluorescent Proteins for Far-Field Fluorescence Nanoscopy," Biophys. J. 95, 2989-2997 (2008).
[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. Schönle, and S. W. Hell, "Fluorescence nanoscopy in whole cells by asnychronous localization of photoswitching emitters," Biophys. J. 93, 3285-3290 (2007).
[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]

Kador, L.

W. E. Moerner and L. Kador, "Optical detection and spectroscopy of single molecules in a solid," Phys. Rev. Lett. 62, 2535-2538 (1989).
[CrossRef] [PubMed]

Keller, R. A.

E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper, "Detection of single fluorescent molecules," Chem. Phys. Lett. 174, 553-557 (1990).
[CrossRef]

Kischner, M. W.

T. L. Hill and M. W. Kischner, "Subunit treadmilling of microtubules or actin in the presence of cellular barriers: possible conversion of chemical free energy into mechanical work," Proc. Natl. Acad. Sci. USA 79, 490-494 (1982).
[CrossRef] [PubMed]

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]

Ko, D.-S.

C. Zander, M. Sauer, K. H. Drexhage, D.-S. Ko, A. Schulz, J. Wolfrum, L. Brand, C. Eggeling, and C. A. M. Seidel, "Detection and characterization of single molecules in aqueous solution," Appl. Phys. B 63, 517-523 (1996).

Kunetsky, R.

J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, "Photochromic Rhodamines Provide Nanoscopy with Optical Sectioning," Angew. Chem. Int. Ed. 46, 6266-6270 (2007).
[CrossRef]

Lacoste, T. D.

T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, "Ultrahigh-resolution multicolor colocalization of single fluorescent probes," Proc. Natl. Acad. Sci. USA 97, 9461-9466 (2000).
[CrossRef] [PubMed]

Lamesch, P.

P. Lamesch, N. Li, S. Milstein, C. Fan, T. Hao, G. Szabo, Z. Hu, K. Venkatesan, G. Bethel, P. Martin, J. Rogers, S. Lawlor, S. McLaren, A. Dricot, H. Borick, M. E. Cusick, J. Vandenhaute, I. Dunham, D. E. Hill, and M. Vidal, "hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes," Genomics 89, 307-315 (2007).
[CrossRef] [PubMed]

Larson, D. R.

R. E. Thompson, D. R. Larson, and W. W. Webb, "Precise nanometer localization analysis for individual fluorescent probes," Biophys. J. 82, 2775-2783 (2002).
[CrossRef] [PubMed]

Lawlor, S.

P. Lamesch, N. Li, S. Milstein, C. Fan, T. Hao, G. Szabo, Z. Hu, K. Venkatesan, G. Bethel, P. Martin, J. Rogers, S. Lawlor, S. McLaren, A. Dricot, H. Borick, M. E. Cusick, J. Vandenhaute, I. Dunham, D. E. Hill, and M. Vidal, "hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes," Genomics 89, 307-315 (2007).
[CrossRef] [PubMed]

Li, N.

P. Lamesch, N. Li, S. Milstein, C. Fan, T. Hao, G. Szabo, Z. Hu, K. Venkatesan, G. Bethel, P. Martin, J. Rogers, S. Lawlor, S. McLaren, A. Dricot, H. Borick, M. E. Cusick, J. Vandenhaute, I. Dunham, D. E. Hill, and M. Vidal, "hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes," Genomics 89, 307-315 (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]

Martin, P.

P. Lamesch, N. Li, S. Milstein, C. Fan, T. Hao, G. Szabo, Z. Hu, K. Venkatesan, G. Bethel, P. Martin, J. Rogers, S. Lawlor, S. McLaren, A. Dricot, H. Borick, M. E. Cusick, J. Vandenhaute, I. Dunham, D. E. Hill, and M. Vidal, "hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes," Genomics 89, 307-315 (2007).
[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]

McLaren, S.

P. Lamesch, N. Li, S. Milstein, C. Fan, T. Hao, G. Szabo, Z. Hu, K. Venkatesan, G. Bethel, P. Martin, J. Rogers, S. Lawlor, S. McLaren, A. Dricot, H. Borick, M. E. Cusick, J. Vandenhaute, I. Dunham, D. E. Hill, and M. Vidal, "hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes," Genomics 89, 307-315 (2007).
[CrossRef] [PubMed]

Medda, R.

M. Bossi, J. Fölling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schönle, and S. W. Hell, "Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species," Nano Lett. 8, 2463-2468 (2008).
[CrossRef] [PubMed]

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell "Fluorescence nanoscopy by ground-state depletion and single-molecule return," Nat. Meth. 5, 943 - 945 (2008).
[CrossRef]

A. Egner, C. Geisler, C. von Middendorff, H. Bock, D. Wenzel, R. Medda, M. Andresen, A.-C. Stiel, S. Jakobs, C. Eggeling, A. Schönle, and S. W. Hell, "Fluorescence nanoscopy in whole cells by asnychronous localization of photoswitching emitters," Biophys. J. 93, 3285-3290 (2007).
[CrossRef] [PubMed]

J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, "Photochromic Rhodamines Provide Nanoscopy with Optical Sectioning," Angew. Chem. Int. Ed. 46, 6266-6270 (2007).
[CrossRef]

Michalet, X.

T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, "Ultrahigh-resolution multicolor colocalization of single fluorescent probes," Proc. Natl. Acad. Sci. USA 97, 9461-9466 (2000).
[CrossRef] [PubMed]

Milstein, S.

P. Lamesch, N. Li, S. Milstein, C. Fan, T. Hao, G. Szabo, Z. Hu, K. Venkatesan, G. Bethel, P. Martin, J. Rogers, S. Lawlor, S. McLaren, A. Dricot, H. Borick, M. E. Cusick, J. Vandenhaute, I. Dunham, D. E. Hill, and M. Vidal, "hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes," Genomics 89, 307-315 (2007).
[CrossRef] [PubMed]

Moerner, W. E.

W. E. Moerner and L. Kador, "Optical detection and spectroscopy of single molecules in a solid," Phys. Rev. Lett. 62, 2535-2538 (1989).
[CrossRef] [PubMed]

Nienhaus, G. U.

J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. Röcker, A. Salih, K.-D. Spindler, and G. U. Nienhaus, "EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion," Proc.Natl.Acad.Sci.USA 101, 15905-15910 (2004).
[CrossRef] [PubMed]

Ogletree, D. F.

T. Ha, T. Enderle, D. F. Ogletree, D. S. Chemla, P. R. Selvin, and S. Weiss, "Probing the interaction between two single molecules: Fluorescence resonance energy transfer between a single donor and a single acceptor," Proc. Natl. Acad. Sci. USA 93, 6264-6268 (1996).
[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]

Orrit, M.

M. Orrit and J. Bernard, "Single pentacene molecules detected by fluorescence excitation in a p-terphenyl crystal," Phys. Rev. Lett. 65, 2716-2719 (1990).
[CrossRef] [PubMed]

Oswald, F.

J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. Röcker, A. Salih, K.-D. Spindler, and G. U. Nienhaus, "EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion," Proc.Natl.Acad.Sci.USA 101, 15905-15910 (2004).
[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]

Pinaud, F.

T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, "Ultrahigh-resolution multicolor colocalization of single fluorescent probes," Proc. Natl. Acad. Sci. USA 97, 9461-9466 (2000).
[CrossRef] [PubMed]

Riedel, D.

J. Fölling, V. Belov, D. Riedel, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell "Fluorescence Nanoscopy with Optical Sectioning by Two-Photon Induced Molecular Switching using Continuous-Wave Lasers," ChemPhysChem 9, 321 - 326 (2008).
[CrossRef] [PubMed]

Röcker, C.

J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. Röcker, A. Salih, K.-D. Spindler, and G. U. Nienhaus, "EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion," Proc.Natl.Acad.Sci.USA 101, 15905-15910 (2004).
[CrossRef] [PubMed]

Rogers, J.

P. Lamesch, N. Li, S. Milstein, C. Fan, T. Hao, G. Szabo, Z. Hu, K. Venkatesan, G. Bethel, P. Martin, J. Rogers, S. Lawlor, S. McLaren, A. Dricot, H. Borick, M. E. Cusick, J. Vandenhaute, I. Dunham, D. E. Hill, and M. Vidal, "hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes," Genomics 89, 307-315 (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, 793-796 (2006).
[CrossRef] [PubMed]

Salih, A.

J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. Röcker, A. Salih, K.-D. Spindler, and G. U. Nienhaus, "EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion," Proc.Natl.Acad.Sci.USA 101, 15905-15910 (2004).
[CrossRef] [PubMed]

Sauer, M.

C. Zander, M. Sauer, K. H. Drexhage, D.-S. Ko, A. Schulz, J. Wolfrum, L. Brand, C. Eggeling, and C. A. M. Seidel, "Detection and characterization of single molecules in aqueous solution," Appl. Phys. B 63, 517-523 (1996).

Schaffer, J.

J. Schaffer, A. Volkmer, C. Eggeling, V. Subramaniam, G. Striker, and C. A. M. Seidel, "Identification of single molecules in aqueous solution by time-resolved fluorescence anisotropy," J. Phys. Chem. A 103, 331-336 (1999).
[CrossRef]

Schilde, J.

A. C. Stiel, M. Andresen, H. Bock, M. Hilbert, J. Schilde, A. Schönle, C. Eggeling, A. Egner, S. W. Hell, and S. Jakobs, "Generation of Monomeric Reversibly Switchable Red Fluorescent Proteins for Far-Field Fluorescence Nanoscopy," Biophys. J. 95, 2989-2997 (2008).
[CrossRef] [PubMed]

Schindler, H.

T. Schmidt, G. J. Schutz, W. Baumgartner, H. J. Gruber, and H. Schindler, "Imaging of single molecule diffusion," Proc. Natl. Acad. Sci. USA 93, 2926-9 (1996).
[CrossRef] [PubMed]

Schmidt, T.

T. Schmidt, G. J. Schutz, W. Baumgartner, H. J. Gruber, and H. Schindler, "Imaging of single molecule diffusion," Proc. Natl. Acad. Sci. USA 93, 2926-9 (1996).
[CrossRef] [PubMed]

Schmitt, F.

J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. Röcker, A. Salih, K.-D. Spindler, and G. U. Nienhaus, "EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion," Proc.Natl.Acad.Sci.USA 101, 15905-15910 (2004).
[CrossRef] [PubMed]

Schönle, A.

M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S. W. Hell, and S. Jakobs, "Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy," Nat. Biotechnol. 26, 1035 - 1040 (2008).
[CrossRef] [PubMed]

A. C. Stiel, M. Andresen, H. Bock, M. Hilbert, J. Schilde, A. Schönle, C. Eggeling, A. Egner, S. W. Hell, and S. Jakobs, "Generation of Monomeric Reversibly Switchable Red Fluorescent Proteins for Far-Field Fluorescence Nanoscopy," Biophys. J. 95, 2989-2997 (2008).
[CrossRef] [PubMed]

M. Bossi, J. Fölling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schönle, and S. W. Hell, "Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species," Nano Lett. 8, 2463-2468 (2008).
[CrossRef] [PubMed]

J. Fölling, V. Belov, D. Riedel, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell "Fluorescence Nanoscopy with Optical Sectioning by Two-Photon Induced Molecular Switching using Continuous-Wave Lasers," ChemPhysChem 9, 321 - 326 (2008).
[CrossRef] [PubMed]

A. Schönle and S. W. Hell "Fluorescence nanoscopy goes multicolor," Nature Biotechnol. 25, 1234-1235 (2007).
[CrossRef]

C. Geisler, A. Schönle, C. von Middendorff, H. Bock, C. Eggeling, A. Egner, and S. W. Hell "Resolution of λ/10 in fluorescence microscopy using fast single molecule photo-switching," Appl. Phys. A 88, 223-226 (2007).
[CrossRef]

J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, "Photochromic Rhodamines Provide Nanoscopy with Optical Sectioning," Angew. Chem. Int. Ed. 46, 6266-6270 (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. Schönle, and S. W. Hell, "Fluorescence nanoscopy in whole cells by asnychronous localization of photoswitching emitters," Biophys. J. 93, 3285-3290 (2007).
[CrossRef] [PubMed]

Schulz, A.

C. Zander, M. Sauer, K. H. Drexhage, D.-S. Ko, A. Schulz, J. Wolfrum, L. Brand, C. Eggeling, and C. A. M. Seidel, "Detection and characterization of single molecules in aqueous solution," Appl. Phys. B 63, 517-523 (1996).

Schutz, G. J.

T. Schmidt, G. J. Schutz, W. Baumgartner, H. J. Gruber, and H. Schindler, "Imaging of single molecule diffusion," Proc. Natl. Acad. Sci. USA 93, 2926-9 (1996).
[CrossRef] [PubMed]

Schwille, P.

K. Bacia and P. Schwille, "A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy," Methods 29, 74-85 (2003).
[CrossRef] [PubMed]

Seidel, C. A. M.

J. Schaffer, A. Volkmer, C. Eggeling, V. Subramaniam, G. Striker, and C. A. M. Seidel, "Identification of single molecules in aqueous solution by time-resolved fluorescence anisotropy," J. Phys. Chem. A 103, 331-336 (1999).
[CrossRef]

C. Zander, M. Sauer, K. H. Drexhage, D.-S. Ko, A. Schulz, J. Wolfrum, L. Brand, C. Eggeling, and C. A. M. Seidel, "Detection and characterization of single molecules in aqueous solution," Appl. Phys. B 63, 517-523 (1996).

Seitzinger, N. K.

E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper, "Detection of single fluorescent molecules," Chem. Phys. Lett. 174, 553-557 (1990).
[CrossRef]

Selvin, P. R.

T. Ha, T. Enderle, D. F. Ogletree, D. S. Chemla, P. R. Selvin, and S. Weiss, "Probing the interaction between two single molecules: Fluorescence resonance energy transfer between a single donor and a single acceptor," Proc. Natl. Acad. Sci. USA 93, 6264-6268 (1996).
[CrossRef] [PubMed]

T. Ha, T. Enderle, D. S. Chemla, P. R. Selvin, and S. Weiss, "Single molecule dynamics studied by polarization modulation," Phys. Rev. Lett. 77, 3979-3982 (1996).
[CrossRef] [PubMed]

Shera, E. B.

E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper, "Detection of single fluorescent molecules," Chem. Phys. Lett. 174, 553-557 (1990).
[CrossRef]

Soper, S. A.

E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper, "Detection of single fluorescent molecules," Chem. Phys. Lett. 174, 553-557 (1990).
[CrossRef]

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]

Soukka, J.

S. W. Hell, J. Soukka, and P. E. Hänninen, "Two- and multiphoton detection as an imaging mode and means of increasing the resolution in far-field light microscopy," Bioimaging 3, 65-69 (1995).
[CrossRef]

Spindler, K.-D.

J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. Röcker, A. Salih, K.-D. Spindler, and G. U. Nienhaus, "EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion," Proc.Natl.Acad.Sci.USA 101, 15905-15910 (2004).
[CrossRef] [PubMed]

Stiel, A. C.

M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S. W. Hell, and S. Jakobs, "Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy," Nat. Biotechnol. 26, 1035 - 1040 (2008).
[CrossRef] [PubMed]

A. C. Stiel, M. Andresen, H. Bock, M. Hilbert, J. Schilde, A. Schönle, C. Eggeling, A. Egner, S. W. Hell, and S. Jakobs, "Generation of Monomeric Reversibly Switchable Red Fluorescent Proteins for Far-Field Fluorescence Nanoscopy," Biophys. J. 95, 2989-2997 (2008).
[CrossRef] [PubMed]

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. Schönle, and S. W. Hell, "Fluorescence nanoscopy in whole cells by asnychronous localization of photoswitching emitters," Biophys. J. 93, 3285-3290 (2007).
[CrossRef] [PubMed]

Striker, G.

J. Schaffer, A. Volkmer, C. Eggeling, V. Subramaniam, G. Striker, and C. A. M. Seidel, "Identification of single molecules in aqueous solution by time-resolved fluorescence anisotropy," J. Phys. Chem. A 103, 331-336 (1999).
[CrossRef]

Subramaniam, V.

J. Schaffer, A. Volkmer, C. Eggeling, V. Subramaniam, G. Striker, and C. A. M. Seidel, "Identification of single molecules in aqueous solution by time-resolved fluorescence anisotropy," J. Phys. Chem. A 103, 331-336 (1999).
[CrossRef]

Szabo, G.

P. Lamesch, N. Li, S. Milstein, C. Fan, T. Hao, G. Szabo, Z. Hu, K. Venkatesan, G. Bethel, P. Martin, J. Rogers, S. Lawlor, S. McLaren, A. Dricot, H. Borick, M. E. Cusick, J. Vandenhaute, I. Dunham, D. E. Hill, and M. Vidal, "hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes," Genomics 89, 307-315 (2007).
[CrossRef] [PubMed]

Thompson, R. E.

R. E. Thompson, D. R. Larson, and W. W. Webb, "Precise nanometer localization analysis for individual fluorescent probes," Biophys. J. 82, 2775-2783 (2002).
[CrossRef] [PubMed]

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[CrossRef]

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P. Lamesch, N. Li, S. Milstein, C. Fan, T. Hao, G. Szabo, Z. Hu, K. Venkatesan, G. Bethel, P. Martin, J. Rogers, S. Lawlor, S. McLaren, A. Dricot, H. Borick, M. E. Cusick, J. Vandenhaute, I. Dunham, D. E. Hill, and M. Vidal, "hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes," Genomics 89, 307-315 (2007).
[CrossRef] [PubMed]

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P. Lamesch, N. Li, S. Milstein, C. Fan, T. Hao, G. Szabo, Z. Hu, K. Venkatesan, G. Bethel, P. Martin, J. Rogers, S. Lawlor, S. McLaren, A. Dricot, H. Borick, M. E. Cusick, J. Vandenhaute, I. Dunham, D. E. Hill, and M. Vidal, "hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes," Genomics 89, 307-315 (2007).
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P. Lamesch, N. Li, S. Milstein, C. Fan, T. Hao, G. Szabo, Z. Hu, K. Venkatesan, G. Bethel, P. Martin, J. Rogers, S. Lawlor, S. McLaren, A. Dricot, H. Borick, M. E. Cusick, J. Vandenhaute, I. Dunham, D. E. Hill, and M. Vidal, "hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes," Genomics 89, 307-315 (2007).
[CrossRef] [PubMed]

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J. Schaffer, A. Volkmer, C. Eggeling, V. Subramaniam, G. Striker, and C. A. M. Seidel, "Identification of single molecules in aqueous solution by time-resolved fluorescence anisotropy," J. Phys. Chem. A 103, 331-336 (1999).
[CrossRef]

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C. Geisler, A. Schönle, C. von Middendorff, H. Bock, C. Eggeling, A. Egner, and S. W. Hell "Resolution of λ/10 in fluorescence microscopy using fast single molecule photo-switching," Appl. Phys. A 88, 223-226 (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. Schönle, and S. W. Hell, "Fluorescence nanoscopy in whole cells by asnychronous localization of photoswitching emitters," Biophys. J. 93, 3285-3290 (2007).
[CrossRef] [PubMed]

Vrij, A.

A. van Blaaderen and A. Vrij, "Synthesis and Characterization of Colloidal Dispersions of Fluorescent, Monodisperse Silica Spheres," Langmuir 8, 2921-2931 (1992).
[CrossRef]

Webb, W. W.

R. E. Thompson, D. R. Larson, and W. W. Webb, "Precise nanometer localization analysis for individual fluorescent probes," Biophys. J. 82, 2775-2783 (2002).
[CrossRef] [PubMed]

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T. D. Lacoste, X. Michalet, F. Pinaud, D. S. Chemla, A. P. Alivisatos, and S. Weiss, "Ultrahigh-resolution multicolor colocalization of single fluorescent probes," Proc. Natl. Acad. Sci. USA 97, 9461-9466 (2000).
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T. Ha, T. Enderle, D. F. Ogletree, D. S. Chemla, P. R. Selvin, and S. Weiss, "Probing the interaction between two single molecules: Fluorescence resonance energy transfer between a single donor and a single acceptor," Proc. Natl. Acad. Sci. USA 93, 6264-6268 (1996).
[CrossRef] [PubMed]

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M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S. W. Hell, and S. Jakobs, "Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy," Nat. Biotechnol. 26, 1035 - 1040 (2008).
[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. Schönle, and S. W. Hell, "Fluorescence nanoscopy in whole cells by asnychronous localization of photoswitching emitters," Biophys. J. 93, 3285-3290 (2007).
[CrossRef] [PubMed]

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J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. Röcker, A. Salih, K.-D. Spindler, and G. U. Nienhaus, "EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion," Proc.Natl.Acad.Sci.USA 101, 15905-15910 (2004).
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Wurm, C. A.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell "Fluorescence nanoscopy by ground-state depletion and single-molecule return," Nat. Meth. 5, 943 - 945 (2008).
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C. Zander, M. Sauer, K. H. Drexhage, D.-S. Ko, A. Schulz, J. Wolfrum, L. Brand, C. Eggeling, and C. A. M. Seidel, "Detection and characterization of single molecules in aqueous solution," Appl. Phys. B 63, 517-523 (1996).

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M. J. Rust, M. Bates, and X. Zhuang, "Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)," Nat. Methods 3, 793-796 (2006).
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Angew. Chem. Int. Ed.

J. Fölling, V. Belov, R. Kunetsky, R. Medda, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell, "Photochromic Rhodamines Provide Nanoscopy with Optical Sectioning," Angew. Chem. Int. Ed. 46, 6266-6270 (2007).
[CrossRef]

Appl. Phys. A

C. Geisler, A. Schönle, C. von Middendorff, H. Bock, C. Eggeling, A. Egner, and S. W. Hell "Resolution of λ/10 in fluorescence microscopy using fast single molecule photo-switching," Appl. Phys. A 88, 223-226 (2007).
[CrossRef]

Appl. Phys. B

C. Zander, M. Sauer, K. H. Drexhage, D.-S. Ko, A. Schulz, J. Wolfrum, L. Brand, C. Eggeling, and C. A. M. Seidel, "Detection and characterization of single molecules in aqueous solution," Appl. Phys. B 63, 517-523 (1996).

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R. E. Thompson, D. R. Larson, and W. W. Webb, "Precise nanometer localization analysis for individual fluorescent probes," Biophys. J. 82, 2775-2783 (2002).
[CrossRef] [PubMed]

A. C. Stiel, M. Andresen, H. Bock, M. Hilbert, J. Schilde, A. Schönle, C. Eggeling, A. Egner, S. W. Hell, and S. Jakobs, "Generation of Monomeric Reversibly Switchable Red Fluorescent Proteins for Far-Field Fluorescence Nanoscopy," Biophys. J. 95, 2989-2997 (2008).
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ChemPhysChem

J. Fölling, V. Belov, D. Riedel, A. Schönle, A. Egner, C. Eggeling, M. Bossi, and S. W. Hell "Fluorescence Nanoscopy with Optical Sectioning by Two-Photon Induced Molecular Switching using Continuous-Wave Lasers," ChemPhysChem 9, 321 - 326 (2008).
[CrossRef] [PubMed]

Genomics

P. Lamesch, N. Li, S. Milstein, C. Fan, T. Hao, G. Szabo, Z. Hu, K. Venkatesan, G. Bethel, P. Martin, J. Rogers, S. Lawlor, S. McLaren, A. Dricot, H. Borick, M. E. Cusick, J. Vandenhaute, I. Dunham, D. E. Hill, and M. Vidal, "hORFeome v3.1: A resource of human open reading frames representing over 10,000 human genes," Genomics 89, 307-315 (2007).
[CrossRef] [PubMed]

J. Phys. Chem. A

J. Schaffer, A. Volkmer, C. Eggeling, V. Subramaniam, G. Striker, and C. A. M. Seidel, "Identification of single molecules in aqueous solution by time-resolved fluorescence anisotropy," J. Phys. Chem. A 103, 331-336 (1999).
[CrossRef]

Langmuir

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[CrossRef]

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M. Bossi, J. Fölling, V. N. Belov, V. P. Boyarskiy, R. Medda, A. Egner, C. Eggeling, A. Schönle, and S. W. Hell, "Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species," Nano Lett. 8, 2463-2468 (2008).
[CrossRef] [PubMed]

Nat. Biotechnol.

M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S. W. Hell, and S. Jakobs, "Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy," Nat. Biotechnol. 26, 1035 - 1040 (2008).
[CrossRef] [PubMed]

Nat. Meth.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell "Fluorescence nanoscopy by ground-state depletion and single-molecule return," Nat. Meth. 5, 943 - 945 (2008).
[CrossRef]

Nat. Methods

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

T. Ha, T. Enderle, D. F. Ogletree, D. S. Chemla, P. R. Selvin, and S. Weiss, "Probing the interaction between two single molecules: Fluorescence resonance energy transfer between a single donor and a single acceptor," Proc. Natl. Acad. Sci. USA 93, 6264-6268 (1996).
[CrossRef] [PubMed]

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J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. Röcker, A. Salih, K.-D. Spindler, and G. U. Nienhaus, "EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion," Proc.Natl.Acad.Sci.USA 101, 15905-15910 (2004).
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Figures (6)

Fig. 1.
Fig. 1.

The microscope setup is essentially a regular wide-field microscope equipped with a fast and sensitive CCD camera for detection of single emitters and a choice of several laser sources and beam optics for switching and excitation with circularly polarized light. Fluorescence is split into the two polarization channels by a polarizing beam splitter (PBS) and imaged onto separate areas (1,2) of the same CCD chip. A more detailed description is given in the text. Labels: (ELD) excitation laser diode, (SLD) switching laser diode, (AOTF) acousto-optical tunable filter, (M) mirror, (P1, P2) polarizer, (DC) dichroic filter, (Q) quarter wave plate, (OBJ) objective lens, (PBS) polarizing beamsplitter, (TL) tube lens and (CCD) EMCCD camera.

Fig. 2.
Fig. 2.

Theoretical two-dimensional anisotropy histogram for (a) fixed molecules and (b) molecules rotating much faster than the typical single molecule fluorescence burst duration calculated as predicted by equations (1) and (2). Depolarization due to focusing through a large aperture lens, detection background and slower rotation speeds were not considered and will lead to broader distributions in experimental histograms.

Fig. 3.
Fig. 3.

Two channel images of single pcRhB molecules in (a) PMMA and (b) mowiol. (c) The two-dimensional anisotropy histogram from 9,185 single molecules reveals that pcRhB is static in PMMA. (d) Contrary the histogram from 50,611 pcRhB molecules in mowiol shows that the labels are rotating faster than the event duration. The molecules where excited at 532nm with an intensity of 24 kW/cm2. Photon threshold n 1=n 2=252. Scale bar 1µm.

Fig. 4.
Fig. 4.

(a) Nanoscale imaging of pcRhB-labeled beads covered by homogeneously pcRhB-stained mowiol. The upper left corner exemplifies the resolution that a conventional diffraction- limited image would achieve. It was created by adding up the fluorescence detected for all single-molecule events. (b) Two-dimensional anisotropy histogram revealing a distribution with contributions from static pcRhB in beads and rotating dyes in mowiol. (c) By assigning events to two channels based on their position in the histogram, the two species can be spatially separated. Events caused by static molecules are displayed in green and events caused by freely rotating molecules are displayed in red. (d) Histogram of the events shown in (c). Imaging parameters: Excitation at 532nm with 24 kW/cm2, T=2 ms, number of frames 30,000, photon threshold n 1=n 2=252. Scale bars 1µm.

Fig. 5.
Fig. 5.

Nanoscale image of the endoplasmic reticulum (ER) of living PtK2 cells labeled with rsCherryRev (a) and the corresponding two-dimensional anisotropy histogram (b) revealing only mobile rsCherryRev proteins, and of ß-actin of living PtK2 cells labeled with tdEosFP (c). The two-dimensional anisotropy histogram of ß-actin-tdEosFP reveals static and free rotating molecules (d). Separate nanoscopy images of immobile (green) and mobile ß-actin-tdEosFP (red) are created by proper assignment of events based on their position in the two-dimensional anisotropy histogram (f). The resolution of a conventional diffraction-limited image is exemplified in the upper triangles of (a) and (c) where the fluorescence detected for all single-molecule events has been added up. Excitation at 560nm with 16 kW/cm2 (a) and 8 kW/cm2 (c), T=2 ms, number of frames 10,000 and 20,000, photon threshold n 1=n 2=252. Scale bars 1µm.

Fig. 6.
Fig. 6.

Two-dimensional anisotropy histograms of live PtK2 cells expressing ß-actin-tdEosFP (a) in a normal culture medium, (b) after 15–25 minutes of incubations with latrunculin B and (c) after 30 minutes of recovery from the toxin. All histograms show events from three cells. The insets show wide-field images of exemplary cells. Photon threshold n 1 =n 2 =252. Scale bars 10µm.

Equations (3)

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

g ( n 1 , n 2 ) = f ( n 1 + n 2 ; N p ) 2 n 2 n 2 ( n 1 + n 2 ) ! ( n 1 ! n 2 ! )
g ( n 1 , n 2 ) = d Ω f ( n 1 , n 2 ; N p ( θ ) ) p 1 ( θ , ϕ ) n 1 p 2 ( θ , ϕ ) n 2 ( n 1 + n 2 ) ! ( n 1 ! n 2 ! )
P n = ( n 2 n 1 ) ( n 1 + n 2 ) .

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