Abstract

Stimulated emission depletion (STED) microscopy achieves diffraction-unlimited resolution in far-field fluorescence microscopy well below 100 nm. As common for (single-lens) far-field microscopy techniques, the lateral resolution is better than the axial sectioning capabilities. Here we present the first implementation of total internal reflection (TIR) illumination into STED microscopy which limits fluorophore excitation to ~70 nm in the vicinity of the cover slip while simultaneously providing ~50 nm lateral resolution. We demonstrate the performance of this new microscope technique with fluorescent bead test samples as well as immuno-stained microtubules. Total internal reflection STED microscopy provides superior axial sectioning capabilities with the potential to reduce photo-bleaching and photo-damage in live cell imaging.

© 2011 OSA

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. S. W. Hell, “Microscopy and its focal switch,” Nat. Methods 6(1), 24–32 (2009).
    [CrossRef] [PubMed]
  2. D. Toomre and J. Bewersdorf, “A new wave of cellular imaging,” Annu. Rev. Cell Dev. Biol. 26(1), 285–314 (2010).
    [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. S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
    [CrossRef] [PubMed]
  5. M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
    [CrossRef] [PubMed]
  6. 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]
  7. 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]
  8. M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U.S.A. 102(49), 17565–17569 (2005).
    [CrossRef] [PubMed]
  9. 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]
  10. J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express 15(6), 3361–3371 (2007).
    [CrossRef] [PubMed]
  11. 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]
  12. L. Meyer, D. Wildanger, R. Medda, A. Punge, S. O. Rizzoli, G. Donnert, and S. W. Hell, “Dual-color STED microscopy at 30-nm focal-plane resolution,” Small 4(8), 1095–1100 (2008).
    [CrossRef] [PubMed]
  13. K. I. Willig, R. R. Kellner, R. Medda, B. Hein, S. Jakobs, and S. W. Hell, “Nanoscale resolution in GFP-based microscopy,” Nat. Methods 3(9), 721–723 (2006).
    [CrossRef] [PubMed]
  14. J. Bückers, D. Wildanger, G. Vicidomini, L. Kastrup, and S. W. Hell, “Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses,” Opt. Express 19(4), 3130–3143 (2011).
    [CrossRef] [PubMed]
  15. M. Dyba, S. Jakobs, and S. W. Hell, “Immunofluorescence stimulated emission depletion microscopy,” Nat. Biotechnol. 21(11), 1303–1304 (2003).
    [CrossRef] [PubMed]
  16. R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
    [CrossRef] [PubMed]
  17. D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol. 89(1), 141–145 (1981).
    [CrossRef] [PubMed]
  18. J. W. M. Chon and M. Gu, “Scanning total internal reflection fluorescence microscopy under one-photon and two-photon excitation: image formation,” Appl. Opt. 43(5), 1063–1071 (2004).
    [CrossRef] [PubMed]
  19. G. Terakado, K. Watanabe, and H. Kano, “Scanning confocal total internal reflection fluorescence microscopy by using radial polarization in the illumination system,” Appl. Opt. 48(6), 1114–1118 (2009).
    [CrossRef]
  20. E. Auksorius, B. R. Boruah, C. Dunsby, P. M. Lanigan, G. Kennedy, M. A. Neil, and P. M. French, “Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging,” Opt. Lett. 33(2), 113–115 (2008).
    [CrossRef] [PubMed]
  21. A. L. Mattheyses and D. Axelrod, “Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence,” J. Biomed. Opt. 11(1), 014006 (2006).
    [CrossRef] [PubMed]
  22. C. A. Wurm, D. Neumann, R. Schmidt, A. Egner, and S. Jakobs, “Sample preparation for STED microscopy,” Methods Mol. Biol. 591, 185–199 (2010).
    [CrossRef] [PubMed]
  23. B. Hein, K. I. Willig, and S. W. Hell, “Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell,” Proc. Natl. Acad. Sci. U.S.A. 105(38), 14271–14276 (2008).
    [CrossRef] [PubMed]
  24. V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
    [CrossRef] [PubMed]

2011

2010

D. Toomre and J. Bewersdorf, “A new wave of cellular imaging,” Annu. Rev. Cell Dev. Biol. 26(1), 285–314 (2010).
[CrossRef] [PubMed]

C. A. Wurm, D. Neumann, R. Schmidt, A. Egner, and S. Jakobs, “Sample preparation for STED microscopy,” Methods Mol. Biol. 591, 185–199 (2010).
[CrossRef] [PubMed]

2009

S. W. Hell, “Microscopy and its focal switch,” Nat. Methods 6(1), 24–32 (2009).
[CrossRef] [PubMed]

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]

G. Terakado, K. Watanabe, and H. Kano, “Scanning confocal total internal reflection fluorescence microscopy by using radial polarization in the illumination system,” Appl. Opt. 48(6), 1114–1118 (2009).
[CrossRef]

2008

E. Auksorius, B. R. Boruah, C. Dunsby, P. M. Lanigan, G. Kennedy, M. A. Neil, and P. M. French, “Stimulated emission depletion microscopy with a supercontinuum source and fluorescence lifetime imaging,” Opt. Lett. 33(2), 113–115 (2008).
[CrossRef] [PubMed]

B. Hein, K. I. Willig, and S. W. Hell, “Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell,” Proc. Natl. Acad. Sci. U.S.A. 105(38), 14271–14276 (2008).
[CrossRef] [PubMed]

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

L. Meyer, D. Wildanger, R. Medda, A. Punge, S. O. Rizzoli, G. Donnert, and S. W. Hell, “Dual-color STED microscopy at 30-nm focal-plane resolution,” Small 4(8), 1095–1100 (2008).
[CrossRef] [PubMed]

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[CrossRef] [PubMed]

2007

2006

A. L. Mattheyses and D. Axelrod, “Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence,” J. Biomed. Opt. 11(1), 014006 (2006).
[CrossRef] [PubMed]

K. I. Willig, R. R. Kellner, R. Medda, B. Hein, S. Jakobs, and S. W. Hell, “Nanoscale resolution in GFP-based microscopy,” Nat. Methods 3(9), 721–723 (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]

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. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

2005

M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U.S.A. 102(49), 17565–17569 (2005).
[CrossRef] [PubMed]

2004

2003

M. Dyba, S. Jakobs, and S. W. Hell, “Immunofluorescence stimulated emission depletion microscopy,” Nat. Biotechnol. 21(11), 1303–1304 (2003).
[CrossRef] [PubMed]

2000

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]

1994

1981

D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol. 89(1), 141–145 (1981).
[CrossRef] [PubMed]

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]

Auksorius, E.

Axelrod, D.

A. L. Mattheyses and D. Axelrod, “Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence,” J. Biomed. Opt. 11(1), 014006 (2006).
[CrossRef] [PubMed]

D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol. 89(1), 141–145 (1981).
[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(10), 793–796 (2006).
[CrossRef] [PubMed]

Betzig, E.

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

Bewersdorf, J.

D. Toomre and J. Bewersdorf, “A new wave of cellular imaging,” Annu. Rev. Cell Dev. Biol. 26(1), 285–314 (2010).
[CrossRef] [PubMed]

Bonifacino, J. S.

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

Boruah, B. R.

Bückers, J.

Chon, J. W. M.

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]

Donnert, G.

L. Meyer, D. Wildanger, R. Medda, A. Punge, S. O. Rizzoli, G. Donnert, and S. W. Hell, “Dual-color STED microscopy at 30-nm focal-plane resolution,” Small 4(8), 1095–1100 (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]

Dunsby, C.

Dyba, M.

M. Dyba, S. Jakobs, and S. W. Hell, “Immunofluorescence stimulated emission depletion microscopy,” Nat. Biotechnol. 21(11), 1303–1304 (2003).
[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]

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]

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. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U.S.A. 102(49), 17565–17569 (2005).
[CrossRef] [PubMed]

Egner, A.

C. A. Wurm, D. Neumann, R. Schmidt, A. Egner, and S. Jakobs, “Sample preparation for STED microscopy,” Methods Mol. Biol. 591, 185–199 (2010).
[CrossRef] [PubMed]

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (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]

Engelhardt, J.

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[CrossRef] [PubMed]

French, P. M.

Girirajan, T. P.

S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

Gu, M.

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]

Hein, B.

B. Hein, K. I. Willig, and S. W. Hell, “Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell,” Proc. Natl. Acad. Sci. U.S.A. 105(38), 14271–14276 (2008).
[CrossRef] [PubMed]

K. I. Willig, R. R. Kellner, R. Medda, B. Hein, S. Jakobs, and S. W. Hell, “Nanoscale resolution in GFP-based microscopy,” Nat. Methods 3(9), 721–723 (2006).
[CrossRef] [PubMed]

Hell, S. W.

J. Bückers, D. Wildanger, G. Vicidomini, L. Kastrup, and S. W. Hell, “Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses,” Opt. Express 19(4), 3130–3143 (2011).
[CrossRef] [PubMed]

S. W. Hell, “Microscopy and its focal switch,” Nat. Methods 6(1), 24–32 (2009).
[CrossRef] [PubMed]

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]

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[CrossRef] [PubMed]

B. Hein, K. I. Willig, and S. W. Hell, “Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell,” Proc. Natl. Acad. Sci. U.S.A. 105(38), 14271–14276 (2008).
[CrossRef] [PubMed]

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

L. Meyer, D. Wildanger, R. Medda, A. Punge, S. O. Rizzoli, G. Donnert, and S. W. Hell, “Dual-color STED microscopy at 30-nm focal-plane resolution,” Small 4(8), 1095–1100 (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]

K. I. Willig, R. R. Kellner, R. Medda, B. Hein, S. Jakobs, and S. W. Hell, “Nanoscale resolution in GFP-based microscopy,” Nat. Methods 3(9), 721–723 (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]

M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U.S.A. 102(49), 17565–17569 (2005).
[CrossRef] [PubMed]

M. Dyba, S. Jakobs, and S. W. Hell, “Immunofluorescence stimulated emission depletion microscopy,” Nat. Biotechnol. 21(11), 1303–1304 (2003).
[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]

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]

Hess, S. T.

S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

Hofmann, M.

M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U.S.A. 102(49), 17565–17569 (2005).
[CrossRef] [PubMed]

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]

Jahn, R.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (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]

Jakobs, S.

C. A. Wurm, D. Neumann, R. Schmidt, A. Egner, and S. Jakobs, “Sample preparation for STED microscopy,” Methods Mol. Biol. 591, 185–199 (2010).
[CrossRef] [PubMed]

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[CrossRef] [PubMed]

K. I. Willig, R. R. Kellner, R. Medda, B. Hein, S. Jakobs, and S. W. Hell, “Nanoscale resolution in GFP-based microscopy,” Nat. Methods 3(9), 721–723 (2006).
[CrossRef] [PubMed]

M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U.S.A. 102(49), 17565–17569 (2005).
[CrossRef] [PubMed]

M. Dyba, S. Jakobs, and S. W. Hell, “Immunofluorescence stimulated emission depletion microscopy,” Nat. Biotechnol. 21(11), 1303–1304 (2003).
[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]

Kamin, D.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

Kano, H.

Kastrup, L.

Keller, J.

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]

Kellner, R. R.

K. I. Willig, R. R. Kellner, R. Medda, B. Hein, S. Jakobs, and S. W. Hell, “Nanoscale resolution in GFP-based microscopy,” Nat. Methods 3(9), 721–723 (2006).
[CrossRef] [PubMed]

Kennedy, G.

Klar, T. A.

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

Lanigan, P. M.

Lauterbach, M. A.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
[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(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]

Mason, M. D.

S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

Mattheyses, A. L.

A. L. Mattheyses and D. Axelrod, “Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence,” J. Biomed. Opt. 11(1), 014006 (2006).
[CrossRef] [PubMed]

Medda, R.

L. Meyer, D. Wildanger, R. Medda, A. Punge, S. O. Rizzoli, G. Donnert, and S. W. Hell, “Dual-color STED microscopy at 30-nm focal-plane resolution,” Small 4(8), 1095–1100 (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]

K. I. Willig, R. R. Kellner, R. Medda, B. Hein, S. Jakobs, and S. W. Hell, “Nanoscale resolution in GFP-based microscopy,” Nat. Methods 3(9), 721–723 (2006).
[CrossRef] [PubMed]

Meyer, L.

L. Meyer, D. Wildanger, R. Medda, A. Punge, S. O. Rizzoli, G. Donnert, and S. W. Hell, “Dual-color STED microscopy at 30-nm focal-plane resolution,” Small 4(8), 1095–1100 (2008).
[CrossRef] [PubMed]

Neil, M. A.

Neumann, D.

C. A. Wurm, D. Neumann, R. Schmidt, A. Egner, and S. Jakobs, “Sample preparation for STED microscopy,” Methods Mol. Biol. 591, 185–199 (2010).
[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]

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]

Punge, A.

L. Meyer, D. Wildanger, R. Medda, A. Punge, S. O. Rizzoli, G. Donnert, and S. W. Hell, “Dual-color STED microscopy at 30-nm focal-plane resolution,” Small 4(8), 1095–1100 (2008).
[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]

Rizzoli, S. O.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

L. Meyer, D. Wildanger, R. Medda, A. Punge, S. O. Rizzoli, G. Donnert, and S. W. Hell, “Dual-color STED microscopy at 30-nm focal-plane resolution,” Small 4(8), 1095–1100 (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]

Rust, M. J.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

Schmidt, R.

C. A. Wurm, D. Neumann, R. Schmidt, A. Egner, and S. Jakobs, “Sample preparation for STED microscopy,” Methods Mol. Biol. 591, 185–199 (2010).
[CrossRef] [PubMed]

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[CrossRef] [PubMed]

Schönle, A.

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]

Terakado, G.

Toomre, D.

D. Toomre and J. Bewersdorf, “A new wave of cellular imaging,” Annu. Rev. Cell Dev. Biol. 26(1), 285–314 (2010).
[CrossRef] [PubMed]

Vicidomini, G.

Watanabe, K.

Westphal, V.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

Wichmann, J.

Wildanger, D.

J. Bückers, D. Wildanger, G. Vicidomini, L. Kastrup, and S. W. Hell, “Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses,” Opt. Express 19(4), 3130–3143 (2011).
[CrossRef] [PubMed]

L. Meyer, D. Wildanger, R. Medda, A. Punge, S. O. Rizzoli, G. Donnert, and S. W. Hell, “Dual-color STED microscopy at 30-nm focal-plane resolution,” Small 4(8), 1095–1100 (2008).
[CrossRef] [PubMed]

Willig, K. I.

B. Hein, K. I. Willig, and S. W. Hell, “Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell,” Proc. Natl. Acad. Sci. U.S.A. 105(38), 14271–14276 (2008).
[CrossRef] [PubMed]

K. I. Willig, R. R. Kellner, R. Medda, B. Hein, S. Jakobs, and S. W. Hell, “Nanoscale resolution in GFP-based microscopy,” Nat. Methods 3(9), 721–723 (2006).
[CrossRef] [PubMed]

Wurm, C. A.

C. A. Wurm, D. Neumann, R. Schmidt, A. Egner, and S. Jakobs, “Sample preparation for STED microscopy,” Methods Mol. Biol. 591, 185–199 (2010).
[CrossRef] [PubMed]

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[CrossRef] [PubMed]

Zhuang, X.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

Annu. Rev. Cell Dev. Biol.

D. Toomre and J. Bewersdorf, “A new wave of cellular imaging,” Annu. Rev. Cell Dev. Biol. 26(1), 285–314 (2010).
[CrossRef] [PubMed]

Appl. Opt.

Biophys. J.

S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

J. Biomed. Opt.

A. L. Mattheyses and D. Axelrod, “Direct measurement of the evanescent field profile produced by objective-based total internal reflection fluorescence,” J. Biomed. Opt. 11(1), 014006 (2006).
[CrossRef] [PubMed]

J. Cell Biol.

D. Axelrod, “Cell-substrate contacts illuminated by total internal reflection fluorescence,” J. Cell Biol. 89(1), 141–145 (1981).
[CrossRef] [PubMed]

Methods Mol. Biol.

C. A. Wurm, D. Neumann, R. Schmidt, A. Egner, and S. Jakobs, “Sample preparation for STED microscopy,” Methods Mol. Biol. 591, 185–199 (2010).
[CrossRef] [PubMed]

Nat. Biotechnol.

M. Dyba, S. Jakobs, and S. W. Hell, “Immunofluorescence stimulated emission depletion microscopy,” Nat. Biotechnol. 21(11), 1303–1304 (2003).
[CrossRef] [PubMed]

Nat. Methods

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[CrossRef] [PubMed]

S. W. Hell, “Microscopy and its focal switch,” Nat. Methods 6(1), 24–32 (2009).
[CrossRef] [PubMed]

K. I. Willig, R. R. Kellner, R. Medda, B. Hein, S. Jakobs, and S. W. Hell, “Nanoscale resolution in GFP-based microscopy,” Nat. Methods 3(9), 721–723 (2006).
[CrossRef] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

Nat. Photonics

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. Express

Opt. Lett.

Proc. Natl. Acad. Sci. U.S.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]

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]

M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U.S.A. 102(49), 17565–17569 (2005).
[CrossRef] [PubMed]

B. Hein, K. I. Willig, and S. W. Hell, “Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell,” Proc. Natl. Acad. Sci. U.S.A. 105(38), 14271–14276 (2008).
[CrossRef] [PubMed]

Science

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

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

Small

L. Meyer, D. Wildanger, R. Medda, A. Punge, S. O. Rizzoli, G. Donnert, and S. W. Hell, “Dual-color STED microscopy at 30-nm focal-plane resolution,” Small 4(8), 1095–1100 (2008).
[CrossRef] [PubMed]

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (4)

Fig. 1
Fig. 1

Schematic of TIRF-STED setup. FI – Faraday Isolator; λ/2 – half-wave plate; GLP – Glen Laser Polarizer; GB – Glas block; DS – delay stage; AOM – Acousto-optical modulator; PC- photonic crystal fiber; F1-3 bandpass filters; PMF – polarization-maintaining single mode fibers; PM – phase mask; PBS – polarizing beam splitter cubes; DM1/DM2 – dichroic mirrors; OBJ – objective lens; xyz – 3-axes piezo sample stage; SWP – segmented wave plate; λ/4 – quarter-wave plate; BB – removable beam blocks; AA – annular aperture; BS – beam splitter cube; MMF – multimode fiber; APD – single-photon counting avalanche photodiode. Not shown are a few additional mirrors and the microscope stand.

Fig. 2
Fig. 2

Conventional and evanescent field confocal excitation of a 6 µm bead. xz-scans through the center of the same surface-labeled fluorescent bead with conventional (A) and TIRF (B) excitation. An intensity profile analysis (C) of xy-images taken in the plane of the cover slip surface (D) allows the determination of the evanescent field depth.

Fig. 3
Fig. 3

Lateral resolution of the different imaging modes as determined from 40 nm bead images. A, B, D, E – xy-images of fluorescent beads in water sticking to the cover slip. C – Line profiles through the beads as denoted by the white boxes in (A) and (B). F - Line profiles through the beads as denoted by the white boxes in (D) and (E). Profiles were averaged in the y-direction over the width of the boxes.

Fig. 4
Fig. 4

Biological application of TIRF-STED microscopy. Immuno-stained microtubules in a PtK2 cell were imaged in conventional confocal (A) and STED (B) mode as well as in confocal TIRF (D) and TIRF-STED (E) mode. (C) and (F) show linearly deconvolved versions of (B) and (E), respectively. The arrow heads highlight tubules that are well visible in STED mode but nearly disappear in TIRF-STED mode indicating their position deeper in the cell outside the evanescent excitation field. G - Overlay of the deconvolved STED (shown in green) and TIRF-STED (shown in purple) images. H - profile as depicted by the box shown in (G). Data has been averaged along the short side of the box.

Equations (1)

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

d = r r 2 ( a / 2 ) 2

Metrics