Abstract

Stimulated emission depletion (STED) microscopy usually employs a scanning excitation beam that is superimposed by a donut-shaped STED beam for keeping the fluorophores at the periphery of the excitation spot dark. Here, we introduce a simple birefringent device that produces a donut-shaped focal spot with suitable polarization for STED, while leaving the excitation spot virtually intact. The device instantly converts a scanning (confocal) microscope with a co-aligned STED beam into a full-blown STED microscope. The donut can be adapted to reveal, through the resulting fluorescence image, the orientation of fluorophores in the sample, thus directly providing subdiffraction resolution images of molecular orientation.

© 2010 OSA

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  1. S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 780–782 (1994).
    [CrossRef] [PubMed]
  2. T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
    [CrossRef] [PubMed]
  3. 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(5793), 1642–1645 (2006).
    [CrossRef] [PubMed]
  4. M. J. Rust, M. Bates, and X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
    [CrossRef] [PubMed]
  5. M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(Pt 2), 82–87 (2000).
    [CrossRef] [PubMed]
  6. G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
    [CrossRef] [PubMed]
  7. V. Westphal and S. W. Hell, “Nanoscale resolution in the focal plane of an optical microscope,” Phys. Rev. Lett. 94(14), 143903 (2005).
    [CrossRef] [PubMed]
  8. E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “Sted microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
    [CrossRef]
  9. D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. 236(1), 35–43 (2009).
    [CrossRef] [PubMed]
  10. S. W. Hell, “Far-field optical nanoscopy,” Science 316(5828), 1153–1158 (2007).
    [CrossRef] [PubMed]
  11. J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express 15(6), 3361–3371 (2007).
    [CrossRef] [PubMed]
  12. B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16(6), 4154–4162 (2008).
    [CrossRef] [PubMed]
  13. D. Wildanger, E. Rittweger, L. Kastrup, and S. W. Hell, “STED microscopy with a supercontinuum laser source,” Opt. Express 16(13), 9614–9621 (2008).
    [CrossRef] [PubMed]
  14. N. Bokor, Y. Iketaki, T. Watanabe, and M. Fujii, “Compact fluorescence depletion microscope system using an integrated optical element,” Opt. Commun. 281(7), 1850–1854 (2008).
    [CrossRef]
  15. R. Menon, P. Rogge, and H.-Y. Tsai, “Design of diffractive lenses that generate optical nulls without phase singularities,” J. Opt. Soc. Am. A 26(2), 297–304 (2009).
    [CrossRef]
  16. D. Wildanger, J. Bückers, V. Westphal, S. W. Hell, and L. Kastrup, “A STED microscope aligned by design,” Opt. Express 17(18), 16100–16110 (2009).
    [CrossRef] [PubMed]
  17. G. Machavariani, Y. Lumer, I. Moshe, A. Meir, and S. Jackel, “Efficient extracavity generation of radially and azimuthally polarized beams,” Opt. Lett. 32(11), 1468–1470 (2007).
    [CrossRef] [PubMed]
  18. M. Dyba, J. Keller, and S. W. Hell, “Phase filter enhanced STED-4Pi fluorescence microscopy: theory and experiment,” N. J. Phys. 7, 134 (2005).
    [CrossRef]
  19. N. Bokor, Y. Iketaki, T. Watanabe, K. Daigoku, N. Davidson, and M. Fujii, “On polarization effects in fluorescence depletion microscopy,” Opt. Commun. 272(1), 263–268 (2007).
    [CrossRef]
  20. V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, “Laser-diode-stimulated emission depletion microscopy,” Appl. Phys. Lett. 82(18), 3125–3127 (2003).
    [CrossRef]
  21. P. Török and P. Munro, “The use of Gauss-Laguerre vector beams in STED microscopy,” Opt. Express 12(15), 3605–3617 (2004).
    [CrossRef] [PubMed]
  22. I. Testa, A. Schönle, C. von Middendorff, C. Geisler, R. Medda, C. A. Wurm, A. C. Stiel, S. Jakobs, M. Bossi, C. Eggeling, S. W. Hell, and A. Egner, “Nanoscale separation of molecular species based on their rotational mobility,” Opt. Express 16(25), 21093–21104 (2008).
    [CrossRef] [PubMed]
  23. M. A. Lieb, J. M. Zavislan, and L. Novotny, “Single-molecule orientations determined by direct emission pattern imaging,” J. Opt. Soc. Am. B 21(6), 1210–1215 (2004).
    [CrossRef]
  24. M. Böhmer and J. Enderlein, “Orientation imaging of single molecules by wide-field epifluorescence microscopy,” Opt. Soc. Am. B 20(3), 554–559 (2003).
    [CrossRef]
  25. P. Dedecker, B. Muls, J. Hofkens, J. Enderlein, and J. I. Hotta, “Orientational effects in the excitation and de-excitation of single molecules interacting with donut-mode laser beams,” Opt. Express 15(6), 3372–3383 (2007).
    [CrossRef] [PubMed]
  26. D. Patra, I. Gregor, and J. Enderlein, “Image Analysis of Defocused Single-Molecule Images for Three-Dimensional Molecule Orientation Studies,” J. Phys. Chem. A 108(33), 6836 (2004).
    [CrossRef]
  27. T. Ha, T. Enderle, S. Chemla, R. Selvin, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77(19), 3979–3982 (1996).
    [CrossRef] [PubMed]
  28. B. Sick, B. Hecht, and L. Novotny, “Orientational imaging of single molecules by annular illumination,” Phys. Rev. Lett. 85(21), 4482–4485 (2000).
    [CrossRef] [PubMed]

2009 (4)

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “Sted microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[CrossRef]

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. 236(1), 35–43 (2009).
[CrossRef] [PubMed]

R. Menon, P. Rogge, and H.-Y. Tsai, “Design of diffractive lenses that generate optical nulls without phase singularities,” J. Opt. Soc. Am. A 26(2), 297–304 (2009).
[CrossRef]

D. Wildanger, J. Bückers, V. Westphal, S. W. Hell, and L. Kastrup, “A STED microscope aligned by design,” Opt. Express 17(18), 16100–16110 (2009).
[CrossRef] [PubMed]

2008 (4)

2007 (5)

2006 (3)

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(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

M. J. Rust, M. Bates, and X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
[CrossRef] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

2005 (2)

V. Westphal and S. W. Hell, “Nanoscale resolution in the focal plane of an optical microscope,” Phys. Rev. Lett. 94(14), 143903 (2005).
[CrossRef] [PubMed]

M. Dyba, J. Keller, and S. W. Hell, “Phase filter enhanced STED-4Pi fluorescence microscopy: theory and experiment,” N. J. Phys. 7, 134 (2005).
[CrossRef]

2004 (3)

2003 (2)

M. Böhmer and J. Enderlein, “Orientation imaging of single molecules by wide-field epifluorescence microscopy,” Opt. Soc. Am. B 20(3), 554–559 (2003).
[CrossRef]

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

2000 (3)

B. Sick, B. Hecht, and L. Novotny, “Orientational imaging of single molecules by annular illumination,” Phys. Rev. Lett. 85(21), 4482–4485 (2000).
[CrossRef] [PubMed]

M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(Pt 2), 82–87 (2000).
[CrossRef] [PubMed]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

1996 (1)

T. Ha, T. Enderle, S. Chemla, R. Selvin, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77(19), 3979–3982 (1996).
[CrossRef] [PubMed]

1994 (1)

Andrei, M. A.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Bates, M.

M. J. Rust, M. Bates, and X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
[CrossRef] [PubMed]

Betzig, E.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Blanca, C. M.

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

Böhmer, M.

M. Böhmer and J. Enderlein, “Orientation imaging of single molecules by wide-field epifluorescence microscopy,” Opt. Soc. Am. B 20(3), 554–559 (2003).
[CrossRef]

Bokor, N.

N. Bokor, Y. Iketaki, T. Watanabe, and M. Fujii, “Compact fluorescence depletion microscope system using an integrated optical element,” Opt. Commun. 281(7), 1850–1854 (2008).
[CrossRef]

N. Bokor, Y. Iketaki, T. Watanabe, K. Daigoku, N. Davidson, and M. Fujii, “On polarization effects in fluorescence depletion microscopy,” Opt. Commun. 272(1), 263–268 (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(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Bossi, M.

Bückers, J.

Chemla, S.

T. Ha, T. Enderle, S. Chemla, R. Selvin, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77(19), 3979–3982 (1996).
[CrossRef] [PubMed]

Daigoku, K.

N. Bokor, Y. Iketaki, T. Watanabe, K. Daigoku, N. Davidson, and M. Fujii, “On polarization effects in fluorescence depletion microscopy,” Opt. Commun. 272(1), 263–268 (2007).
[CrossRef]

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(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Davidson, N.

N. Bokor, Y. Iketaki, T. Watanabe, K. Daigoku, N. Davidson, and M. Fujii, “On polarization effects in fluorescence depletion microscopy,” Opt. Commun. 272(1), 263–268 (2007).
[CrossRef]

Dedecker, P.

Donnert, G.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Dyba, M.

M. Dyba, J. Keller, and S. W. Hell, “Phase filter enhanced STED-4Pi fluorescence microscopy: theory and experiment,” N. J. Phys. 7, 134 (2005).
[CrossRef]

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

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

Eggeling, C.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “Sted microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[CrossRef]

I. Testa, A. Schönle, C. von Middendorff, C. Geisler, R. Medda, C. A. Wurm, A. C. Stiel, S. Jakobs, M. Bossi, C. Eggeling, S. W. Hell, and A. Egner, “Nanoscale separation of molecular species based on their rotational mobility,” Opt. Express 16(25), 21093–21104 (2008).
[CrossRef] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Egner, A.

I. Testa, A. Schönle, C. von Middendorff, C. Geisler, R. Medda, C. A. Wurm, A. C. Stiel, S. Jakobs, M. Bossi, C. Eggeling, S. W. Hell, and A. Egner, “Nanoscale separation of molecular species based on their rotational mobility,” Opt. Express 16(25), 21093–21104 (2008).
[CrossRef] [PubMed]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

Enderle, T.

T. Ha, T. Enderle, S. Chemla, R. Selvin, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77(19), 3979–3982 (1996).
[CrossRef] [PubMed]

Enderlein, J.

P. Dedecker, B. Muls, J. Hofkens, J. Enderlein, and J. I. Hotta, “Orientational effects in the excitation and de-excitation of single molecules interacting with donut-mode laser beams,” Opt. Express 15(6), 3372–3383 (2007).
[CrossRef] [PubMed]

D. Patra, I. Gregor, and J. Enderlein, “Image Analysis of Defocused Single-Molecule Images for Three-Dimensional Molecule Orientation Studies,” J. Phys. Chem. A 108(33), 6836 (2004).
[CrossRef]

M. Böhmer and J. Enderlein, “Orientation imaging of single molecules by wide-field epifluorescence microscopy,” Opt. Soc. Am. B 20(3), 554–559 (2003).
[CrossRef]

Fujii, M.

N. Bokor, Y. Iketaki, T. Watanabe, and M. Fujii, “Compact fluorescence depletion microscope system using an integrated optical element,” Opt. Commun. 281(7), 1850–1854 (2008).
[CrossRef]

N. Bokor, Y. Iketaki, T. Watanabe, K. Daigoku, N. Davidson, and M. Fujii, “On polarization effects in fluorescence depletion microscopy,” Opt. Commun. 272(1), 263–268 (2007).
[CrossRef]

Geisler, C.

Gregor, I.

D. Patra, I. Gregor, and J. Enderlein, “Image Analysis of Defocused Single-Molecule Images for Three-Dimensional Molecule Orientation Studies,” J. Phys. Chem. A 108(33), 6836 (2004).
[CrossRef]

Gustafsson, M. G. L.

M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(Pt 2), 82–87 (2000).
[CrossRef] [PubMed]

Ha, T.

T. Ha, T. Enderle, S. Chemla, R. Selvin, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77(19), 3979–3982 (1996).
[CrossRef] [PubMed]

Han, K. Y.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “Sted microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[CrossRef]

Harke, B.

Hecht, B.

B. Sick, B. Hecht, and L. Novotny, “Orientational imaging of single molecules by annular illumination,” Phys. Rev. Lett. 85(21), 4482–4485 (2000).
[CrossRef] [PubMed]

Hell, S. W.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “Sted microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[CrossRef]

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. 236(1), 35–43 (2009).
[CrossRef] [PubMed]

D. Wildanger, J. Bückers, V. Westphal, S. W. Hell, and L. Kastrup, “A STED microscope aligned by design,” Opt. Express 17(18), 16100–16110 (2009).
[CrossRef] [PubMed]

I. Testa, A. Schönle, C. von Middendorff, C. Geisler, R. Medda, C. A. Wurm, A. C. Stiel, S. Jakobs, M. Bossi, C. Eggeling, S. W. Hell, and A. Egner, “Nanoscale separation of molecular species based on their rotational mobility,” Opt. Express 16(25), 21093–21104 (2008).
[CrossRef] [PubMed]

D. Wildanger, E. Rittweger, L. Kastrup, and S. W. Hell, “STED microscopy with a supercontinuum laser source,” Opt. Express 16(13), 9614–9621 (2008).
[CrossRef] [PubMed]

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

J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express 15(6), 3361–3371 (2007).
[CrossRef] [PubMed]

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

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

V. Westphal and S. W. Hell, “Nanoscale resolution in the focal plane of an optical microscope,” Phys. Rev. Lett. 94(14), 143903 (2005).
[CrossRef] [PubMed]

M. Dyba, J. Keller, and S. W. Hell, “Phase filter enhanced STED-4Pi fluorescence microscopy: theory and experiment,” N. J. Phys. 7, 134 (2005).
[CrossRef]

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

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 780–782 (1994).
[CrossRef] [PubMed]

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(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Hofkens, J.

Hotta, J. I.

Iketaki, Y.

N. Bokor, Y. Iketaki, T. Watanabe, and M. Fujii, “Compact fluorescence depletion microscope system using an integrated optical element,” Opt. Commun. 281(7), 1850–1854 (2008).
[CrossRef]

N. Bokor, Y. Iketaki, T. Watanabe, K. Daigoku, N. Davidson, and M. Fujii, “On polarization effects in fluorescence depletion microscopy,” Opt. Commun. 272(1), 263–268 (2007).
[CrossRef]

Irvine, S. E.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “Sted microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[CrossRef]

Jackel, S.

Jahn, R.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Jakobs, S.

I. Testa, A. Schönle, C. von Middendorff, C. Geisler, R. Medda, C. A. Wurm, A. C. Stiel, S. Jakobs, M. Bossi, C. Eggeling, S. W. Hell, and A. Egner, “Nanoscale separation of molecular species based on their rotational mobility,” Opt. Express 16(25), 21093–21104 (2008).
[CrossRef] [PubMed]

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

Kastrup, L.

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. 236(1), 35–43 (2009).
[CrossRef] [PubMed]

D. Wildanger, J. Bückers, V. Westphal, S. W. Hell, and L. Kastrup, “A STED microscope aligned by design,” Opt. Express 17(18), 16100–16110 (2009).
[CrossRef] [PubMed]

D. Wildanger, E. Rittweger, L. Kastrup, and S. W. Hell, “STED microscopy with a supercontinuum laser source,” Opt. Express 16(13), 9614–9621 (2008).
[CrossRef] [PubMed]

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

Keller, J.

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

J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express 15(6), 3361–3371 (2007).
[CrossRef] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

M. Dyba, J. Keller, and S. W. Hell, “Phase filter enhanced STED-4Pi fluorescence microscopy: theory and experiment,” N. J. Phys. 7, 134 (2005).
[CrossRef]

Klar, T. A.

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

Lieb, M. A.

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(5793), 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(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Lührmann, R.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Lumer, Y.

Machavariani, G.

Medda, R.

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. 236(1), 35–43 (2009).
[CrossRef] [PubMed]

I. Testa, A. Schönle, C. von Middendorff, C. Geisler, R. Medda, C. A. Wurm, A. C. Stiel, S. Jakobs, M. Bossi, C. Eggeling, S. W. Hell, and A. Egner, “Nanoscale separation of molecular species based on their rotational mobility,” Opt. Express 16(25), 21093–21104 (2008).
[CrossRef] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Meir, A.

Menon, R.

Moshe, I.

Muls, B.

Munro, P.

Novotny, L.

M. A. Lieb, J. M. Zavislan, and L. Novotny, “Single-molecule orientations determined by direct emission pattern imaging,” J. Opt. Soc. Am. B 21(6), 1210–1215 (2004).
[CrossRef]

B. Sick, B. Hecht, and L. Novotny, “Orientational imaging of single molecules by annular illumination,” Phys. Rev. Lett. 85(21), 4482–4485 (2000).
[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(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Patra, D.

D. Patra, I. Gregor, and J. Enderlein, “Image Analysis of Defocused Single-Molecule Images for Three-Dimensional Molecule Orientation Studies,” J. Phys. Chem. A 108(33), 6836 (2004).
[CrossRef]

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(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Rittweger, E.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “Sted microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[CrossRef]

D. Wildanger, E. Rittweger, L. Kastrup, and S. W. Hell, “STED microscopy with a supercontinuum laser source,” Opt. Express 16(13), 9614–9621 (2008).
[CrossRef] [PubMed]

Rizzoli, S. O.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Rogge, P.

Rust, M. J.

M. J. Rust, M. Bates, and X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
[CrossRef] [PubMed]

Schönle, A.

Selvin, R.

T. Ha, T. Enderle, S. Chemla, R. Selvin, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77(19), 3979–3982 (1996).
[CrossRef] [PubMed]

Sick, B.

B. Sick, B. Hecht, and L. Novotny, “Orientational imaging of single molecules by annular illumination,” Phys. Rev. Lett. 85(21), 4482–4485 (2000).
[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(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Stiel, A. C.

Testa, I.

Török, P.

Tsai, H.-Y.

Ullal, C. K.

von Middendorff, C.

Watanabe, T.

N. Bokor, Y. Iketaki, T. Watanabe, and M. Fujii, “Compact fluorescence depletion microscope system using an integrated optical element,” Opt. Commun. 281(7), 1850–1854 (2008).
[CrossRef]

N. Bokor, Y. Iketaki, T. Watanabe, K. Daigoku, N. Davidson, and M. Fujii, “On polarization effects in fluorescence depletion microscopy,” Opt. Commun. 272(1), 263–268 (2007).
[CrossRef]

Weiss, S.

T. Ha, T. Enderle, S. Chemla, R. Selvin, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77(19), 3979–3982 (1996).
[CrossRef] [PubMed]

Westphal, V.

D. Wildanger, J. Bückers, V. Westphal, S. W. Hell, and L. Kastrup, “A STED microscope aligned by design,” Opt. Express 17(18), 16100–16110 (2009).
[CrossRef] [PubMed]

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

V. Westphal and S. W. Hell, “Nanoscale resolution in the focal plane of an optical microscope,” Phys. Rev. Lett. 94(14), 143903 (2005).
[CrossRef] [PubMed]

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

Wichmann, J.

Wildanger, D.

Wurm, C. A.

Zavislan, J. M.

Zhuang, X. W.

M. J. Rust, M. Bates, and X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
[CrossRef] [PubMed]

Appl. Phys. Lett. (1)

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

J. Microsc. (2)

M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(Pt 2), 82–87 (2000).
[CrossRef] [PubMed]

D. Wildanger, R. Medda, L. Kastrup, and S. W. Hell, “A compact STED microscope providing 3D nanoscale resolution,” J. Microsc. 236(1), 35–43 (2009).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A (1)

J. Opt. Soc. Am. B (1)

J. Phys. Chem. A (1)

D. Patra, I. Gregor, and J. Enderlein, “Image Analysis of Defocused Single-Molecule Images for Three-Dimensional Molecule Orientation Studies,” J. Phys. Chem. A 108(33), 6836 (2004).
[CrossRef]

N. J. Phys. (1)

M. Dyba, J. Keller, and S. W. Hell, “Phase filter enhanced STED-4Pi fluorescence microscopy: theory and experiment,” N. J. Phys. 7, 134 (2005).
[CrossRef]

Nat. Methods (1)

M. J. Rust, M. Bates, and X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
[CrossRef] [PubMed]

Nat. Photonics (1)

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “Sted microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[CrossRef]

Opt. Commun. (2)

N. Bokor, Y. Iketaki, T. Watanabe, and M. Fujii, “Compact fluorescence depletion microscope system using an integrated optical element,” Opt. Commun. 281(7), 1850–1854 (2008).
[CrossRef]

N. Bokor, Y. Iketaki, T. Watanabe, K. Daigoku, N. Davidson, and M. Fujii, “On polarization effects in fluorescence depletion microscopy,” Opt. Commun. 272(1), 263–268 (2007).
[CrossRef]

Opt. Express (7)

Opt. Lett. (2)

Opt. Soc. Am. B (1)

M. Böhmer and J. Enderlein, “Orientation imaging of single molecules by wide-field epifluorescence microscopy,” Opt. Soc. Am. B 20(3), 554–559 (2003).
[CrossRef]

Phys. Rev. Lett. (3)

T. Ha, T. Enderle, S. Chemla, R. Selvin, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77(19), 3979–3982 (1996).
[CrossRef] [PubMed]

B. Sick, B. Hecht, and L. Novotny, “Orientational imaging of single molecules by annular illumination,” Phys. Rev. Lett. 85(21), 4482–4485 (2000).
[CrossRef] [PubMed]

V. Westphal and S. W. Hell, “Nanoscale resolution in the focal plane of an optical microscope,” Phys. Rev. Lett. 94(14), 143903 (2005).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. U.S.A. (2)

T. A. Klar, S. Jakobs, M. Dyba, A. Egner, and S. W. Hell, “Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission,” Proc. Natl. Acad. Sci. U.S.A. 97(15), 8206–8210 (2000).
[CrossRef] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A. 103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Science (2)

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(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

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

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Figures (6)

Fig. 1
Fig. 1

Schematic setup of an easySTED microscope using a birefringent beam shaping device directly in the back of the objective lens. Center: principle beam path. Excitation (blue)- and STED-beams (red), as well as emitted fluorescence light (yellow) pass the beam shaping device. Preferably, STED and excitation light are provided by the same source to facilitate intrinsic beam alignment. Left side: Schematic drawing of the beam shaping device, consisting of three wave plates, where the last two wave plates can be combined into a single segmented one, as indicated. Right: polarization state of the excitation- and STED-beams at different stages in the beam shaping device and at two representative points of the wave cycle. The two beams are treated differently due to the chromatic nature of the segmented wave plate. The STED beam will focus to a donut, whereas the excitation beam leaves the device circularly polarized, rendering a normally focused light spot.

Fig. 2
Fig. 2

Effect of the STED beam on a fluorescent molecule with easySTED (a), and with Molecular Orientation STED Microscopy (MOM-STED) (b, c). (a): Polarization distribution in the STED-donut for phases 0 and π / 2 (top row), and the resultant effective STED beam focal spot (STED-PSF) in the second row, taking into account the projection of the STED-field onto the dipole. The effective STED PSFs for ϕ = 0 and ϕ = π / 2 complement each other and lead to high resolution in all directions. (b): with MOM-STED, the resulting effective STED PSF is the same for all ϕ and resolution enhancement is parallel to the dipole only. In (c), a molecule with different orientation is shown; also in this case, the molecule is subject to the STED field only in those regions where the STED-field is parallel to the dipole. It can be seen that with MOM-STED this is always the case in a direction perpendicular to the dipole, regardless of its actual orientation.

Fig. 3
Fig. 3

Beam shaping with a segmented waveplate device. Left: focal intensity distributions of the excitation (blue) and STED (red) beam. Both beams have passed the easySTED beam shaping device simultaneously. The 647nm-STED beam becomes a donut, while the 532nm-excitation beam is focused to a regular spot. Right: line profiles along the direction indicated by the arrows on the left.

Fig. 4
Fig. 4

Resolution enhancement with easySTED. Fluorescent beads imaged in standard confocal mode (left) and with high-resolution easySTED (center). The plot framed green (right) shows a line profile (black) along the arrows indicated in the central easySTED image together with a double Gaussian fit (red). Scale bar = 500 nm; count rates are in counts/200µs dwell time.

Fig. 5
Fig. 5

EasySTED on biological samples. The protein bassoon in hippocampal mouse neurons observed in standard confocal mode (left). In contrast, easySTED (center) reveals detail unobservable in the confocal image. Both images show raw data. Right: profiles along the lines indicated by arrows in the easySTED image together with Gaussian fits reveal a resolution below 40nm. Scale bars are 1µm, count rates are in counts/200µs dwell time.

Fig. 6
Fig. 6

Molecular Orientation Microscopy (MOM) by STED on nile red fluorescent beads bleached down to almost single molecule level. The resolution with STED is enhanced only in directions perpendicular to the dipole of the molecule, as is evident from the magnification on the right. The molecules being mostly vertically aligned is a result of the linearly polarized excitation beam that preferably acts on molecular transition dipoles along this direction. Green arrow: three molecules can be separated even in a sub-STED focal volume solely by the fact that their resulting spot differs in orientation. Scale bars are 500nm, count rates are in counts/1000µs dwell time.

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