Abstract

Super-resolved optical microscopy using stimulated emission depletion (STED) is now a mature method for imaging fluorescent samples at scales beyond the diffraction limit. Nevertheless the practical implementation of STED microscopy is complex and costly, especially since it requires laser beams with different wavelengths for excitation and depletion. In this paper, we propose using a single wavelength to induce both processes. We studied stimulated emission depletion of 4-dicyanomethylene-2-methyl-6-p-dimethylaminostyryl-4H-pyran (DCM) dye with a laser delivering a single wavelength in the near infrared. Fluorescence was excited by two photon absorption with a femtosecond pulse, then depleted by one photon stimulated emission with a stretched pulse. Time-resolved fluorescence decay measurements were performed to determine the depletion efficiency and to prove that fluorescence quenching is not affected by side effects. Numerical simulations show that this method can be applied to super-resolved microscopy.

© 2011 OSA

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    [CrossRef]
  28. E. Rittweger, B. R. Rankin, V. Westphal, and S. W. Hell, “Fluorescence depletion mechanisms in super-resolving STED microscopy,” Chem. Phys. Lett. 442(4-6), 483–487 (2007).
    [CrossRef]
  29. P. R. Hammond, “Laser dye DCM, its spectral properties, synthesis and comparison with other dyes in the red,” Opt. Commun. 29(3), 331–333 (1979).
    [CrossRef]
  30. A. J. Bain, R. J. Marsh, D. A. Armoogum, O. Mongin, L. Porrès, and M. Blanchard-Desce, “Time-resolved stimulated emission depletion in two-photon excited states,” Biochem. Soc. Trans. 31(5), 1047–1051 (2003).
    [CrossRef] [PubMed]
  31. M. Dyba, T. A. Klar, S. Jakobs, and S. W. Hell, “Ultrafast dynamics microscopy,” Appl. Phys. Lett. 77(4), 597–599 (2000).
    [CrossRef]
  32. T. A. Klar and S. W. Hell, “Subdiffraction resolution in far-field fluorescence microscopy,” Opt. Lett. 24(14), 954–956 (1999).
    [CrossRef] [PubMed]
  33. X. Guo and A. Xia, “Ultrafast excited states relaxation dynamics in solution investigated by stimulated emission from a styryl dye,” J. Lumin. 122–123, 532–535 (2007).
    [CrossRef]
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    [CrossRef] [PubMed]

2010 (2)

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and S. W. Hell, “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

K. D. Piatkevich, J. Hulit, O. M. Subach, B. Wu, A. Abdulla, J. E. Segall, and V. V. Verkhusha, “Monomeric red fluorescent proteins with a large Stokes shift,” Proc. Natl. Acad. Sci. U.S.A. 107(12), 5369–5374 (2010).
[CrossRef] [PubMed]

2009 (3)

G. Moneron and S. W. Hell, “Two-photon excitation STED microscopy,” Opt. Express 17(17), 14567–14573 (2009).
[CrossRef] [PubMed]

J. B. Ding, K. T. Takasaki, and B. L. Sabatini, “Supraresolution imaging in brain slices using stimulated-emission depletion two-photon laser scanning microscopy,” Neuron 63(4), 429–437 (2009).
[CrossRef] [PubMed]

Q. Li, S. S. Wu, and K. C. Chou, “Subdiffraction-limit two-photon fluorescence microscopy for GFP-tagged cell imaging,” Biophys. J. 97(12), 3224–3228 (2009).
[CrossRef] [PubMed]

2008 (3)

2007 (3)

X. Guo and A. Xia, “Ultrafast excited states relaxation dynamics in solution investigated by stimulated emission from a styryl dye,” J. Lumin. 122–123, 532–535 (2007).
[CrossRef]

E. Rittweger, B. R. Rankin, V. Westphal, and S. W. Hell, “Fluorescence depletion mechanisms in super-resolving STED microscopy,” Chem. Phys. Lett. 442(4-6), 483–487 (2007).
[CrossRef]

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[CrossRef] [PubMed]

2006 (3)

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]

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

2005 (1)

M. G. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A. 102(37), 13081–13086 (2005).
[CrossRef] [PubMed]

2003 (2)

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]

A. J. Bain, R. J. Marsh, D. A. Armoogum, O. Mongin, L. Porrès, and M. Blanchard-Desce, “Time-resolved stimulated emission depletion in two-photon excited states,” Biochem. Soc. Trans. 31(5), 1047–1051 (2003).
[CrossRef] [PubMed]

2002 (3)

R. Heintzmann, T. M. Jovin, and C. Cremer, “Saturated patterned excitation microscopy--a concept for optical resolution improvement,” J. Opt. Soc. Am. A 19(8), 1599–1609 (2002).
[CrossRef] [PubMed]

R. J. Marsh, D. A. Armoogum, and A. J. Bain, “Stimulated emission depletion of two-photon excited states,” Chem. Phys. Lett. 366(3-4), 398–405 (2002).
[CrossRef]

S. W. Hell, “Increasing the resolution of far-field fluorescence light microscopy by point-spread-function engineering,” Top. Fluoresc. Spectrosc. 5, 361–426 (2002).
[CrossRef]

2000 (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]

M. Dyba, T. A. Klar, S. Jakobs, and S. W. Hell, “Ultrafast dynamics microscopy,” Appl. Phys. Lett. 77(4), 597–599 (2000).
[CrossRef]

1999 (1)

1996 (1)

1995 (1)

S. W. Hell and M. Kroug, “Ground-state-depletion fluorscence microscopy: A concept for breaking the diffraction resolution limit,” Appl. Phys. B 60(5), 495–497 (1995).
[CrossRef]

1994 (3)

J. Kuśba, V. Bogdanov, I. Gryczynski, and J. R. Lakowicz, “Theory of light quenching: effects of fluorescence polarization, intensity, and anisotropy decays,” Biophys. J. 67(5), 2024–2040 (1994).
[CrossRef] [PubMed]

I. Gryczynski, J. Kuśba, V. Bogdanov, and J. R. Lakowicz, “Quenching of fluorescence by light: A new method to control the excited-state lifetimes and orientations of fluorophores,” J. Fluoresc. 4(1), 103–109 (1994).
[CrossRef]

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]

1993 (1)

I. Gryczynski, V. Bogdanov, and J. R. Lakowicz, “Light quenching of tetraphenylbutadiene fluorescence observed during two-photon excitation,” J. Fluoresc. 3(2), 85–92 (1993).
[CrossRef]

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

1985 (1)

J. M. Drake, M. L. Lesiecki, and D. M. Camaioni, “Photophysics and cis-trans isomerization of DCM,” Chem. Phys. Lett. 113(6), 530–534 (1985).
[CrossRef]

1984 (1)

M. Lesiecki, F. Asmar, J. M. Drake, and D. M. Camaioni, “Photoproperties of DCM,” J. Lumin. 31–32, 546–548 (1984).
[CrossRef]

1979 (1)

P. R. Hammond, “Laser dye DCM, its spectral properties, synthesis and comparison with other dyes in the red,” Opt. Commun. 29(3), 331–333 (1979).
[CrossRef]

Abdulla, A.

K. D. Piatkevich, J. Hulit, O. M. Subach, B. Wu, A. Abdulla, J. E. Segall, and V. V. Verkhusha, “Monomeric red fluorescent proteins with a large Stokes shift,” Proc. Natl. Acad. Sci. U.S.A. 107(12), 5369–5374 (2010).
[CrossRef] [PubMed]

Armoogum, D. A.

A. J. Bain, R. J. Marsh, D. A. Armoogum, O. Mongin, L. Porrès, and M. Blanchard-Desce, “Time-resolved stimulated emission depletion in two-photon excited states,” Biochem. Soc. Trans. 31(5), 1047–1051 (2003).
[CrossRef] [PubMed]

R. J. Marsh, D. A. Armoogum, and A. J. Bain, “Stimulated emission depletion of two-photon excited states,” Chem. Phys. Lett. 366(3-4), 398–405 (2002).
[CrossRef]

Asmar, F.

M. Lesiecki, F. Asmar, J. M. Drake, and D. M. Camaioni, “Photoproperties of DCM,” J. Lumin. 31–32, 546–548 (1984).
[CrossRef]

Auksorius, E.

Bain, A. J.

A. J. Bain, R. J. Marsh, D. A. Armoogum, O. Mongin, L. Porrès, and M. Blanchard-Desce, “Time-resolved stimulated emission depletion in two-photon excited states,” Biochem. Soc. Trans. 31(5), 1047–1051 (2003).
[CrossRef] [PubMed]

R. J. Marsh, D. A. Armoogum, and A. J. Bain, “Stimulated emission depletion of two-photon excited states,” Chem. Phys. Lett. 366(3-4), 398–405 (2002).
[CrossRef]

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]

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]

Blanchard-Desce, M.

A. J. Bain, R. J. Marsh, D. A. Armoogum, O. Mongin, L. Porrès, and M. Blanchard-Desce, “Time-resolved stimulated emission depletion in two-photon excited states,” Biochem. Soc. Trans. 31(5), 1047–1051 (2003).
[CrossRef] [PubMed]

Bogdanov, V.

J. Kuśba, V. Bogdanov, I. Gryczynski, and J. R. Lakowicz, “Theory of light quenching: effects of fluorescence polarization, intensity, and anisotropy decays,” Biophys. J. 67(5), 2024–2040 (1994).
[CrossRef] [PubMed]

I. Gryczynski, J. Kuśba, V. Bogdanov, and J. R. Lakowicz, “Quenching of fluorescence by light: A new method to control the excited-state lifetimes and orientations of fluorophores,” J. Fluoresc. 4(1), 103–109 (1994).
[CrossRef]

I. Gryczynski, V. Bogdanov, and J. R. Lakowicz, “Light quenching of tetraphenylbutadiene fluorescence observed during two-photon excitation,” J. Fluoresc. 3(2), 85–92 (1993).
[CrossRef]

Bonhoeffer, T.

U. V. Nägerl, K. I. Willig, B. Hein, S. W. Hell, and T. Bonhoeffer, “Live-cell imaging of dendritic spines by STED microscopy,” Proc. Natl. Acad. Sci. U.S.A. 105(48), 18982–18987 (2008).
[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.

Camaioni, D. M.

J. M. Drake, M. L. Lesiecki, and D. M. Camaioni, “Photophysics and cis-trans isomerization of DCM,” Chem. Phys. Lett. 113(6), 530–534 (1985).
[CrossRef]

M. Lesiecki, F. Asmar, J. M. Drake, and D. M. Camaioni, “Photoproperties of DCM,” J. Lumin. 31–32, 546–548 (1984).
[CrossRef]

Chou, K. C.

Q. Li, S. S. Wu, and K. C. Chou, “Subdiffraction-limit two-photon fluorescence microscopy for GFP-tagged cell imaging,” Biophys. J. 97(12), 3224–3228 (2009).
[CrossRef] [PubMed]

Cremer, C.

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]

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Ding, J. B.

J. B. Ding, K. T. Takasaki, and B. L. Sabatini, “Supraresolution imaging in brain slices using stimulated-emission depletion two-photon laser scanning microscopy,” Neuron 63(4), 429–437 (2009).
[CrossRef] [PubMed]

Drake, J. M.

J. M. Drake, M. L. Lesiecki, and D. M. Camaioni, “Photophysics and cis-trans isomerization of DCM,” Chem. Phys. Lett. 113(6), 530–534 (1985).
[CrossRef]

M. Lesiecki, F. Asmar, J. M. Drake, and D. M. Camaioni, “Photoproperties of DCM,” J. Lumin. 31–32, 546–548 (1984).
[CrossRef]

Dunsby, C.

Dyba, 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]

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. Dyba, T. A. Klar, S. Jakobs, and S. W. Hell, “Ultrafast dynamics microscopy,” Appl. Phys. Lett. 77(4), 597–599 (2000).
[CrossRef]

Egner, 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]

French, P. M. W.

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(11), 4258–4272 (2006).
[CrossRef] [PubMed]

Gryczynski, I.

I. Gryczynski, J. Kuśba, V. Bogdanov, and J. R. Lakowicz, “Quenching of fluorescence by light: A new method to control the excited-state lifetimes and orientations of fluorophores,” J. Fluoresc. 4(1), 103–109 (1994).
[CrossRef]

J. Kuśba, V. Bogdanov, I. Gryczynski, and J. R. Lakowicz, “Theory of light quenching: effects of fluorescence polarization, intensity, and anisotropy decays,” Biophys. J. 67(5), 2024–2040 (1994).
[CrossRef] [PubMed]

I. Gryczynski, V. Bogdanov, and J. R. Lakowicz, “Light quenching of tetraphenylbutadiene fluorescence observed during two-photon excitation,” J. Fluoresc. 3(2), 85–92 (1993).
[CrossRef]

Guo, X.

X. Guo and A. Xia, “Ultrafast excited states relaxation dynamics in solution investigated by stimulated emission from a styryl dye,” J. Lumin. 122–123, 532–535 (2007).
[CrossRef]

Gustafsson, M. G.

M. G. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A. 102(37), 13081–13086 (2005).
[CrossRef] [PubMed]

Hammond, P. R.

P. R. Hammond, “Laser dye DCM, its spectral properties, synthesis and comparison with other dyes in the red,” Opt. Commun. 29(3), 331–333 (1979).
[CrossRef]

Harke, B.

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[CrossRef] [PubMed]

Hein, B.

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and S. W. Hell, “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

U. V. Nägerl, K. I. Willig, B. Hein, S. W. Hell, and T. Bonhoeffer, “Live-cell imaging of dendritic spines by STED microscopy,” Proc. Natl. Acad. Sci. U.S.A. 105(48), 18982–18987 (2008).
[CrossRef] [PubMed]

Heintzmann, R.

Hell, S. W.

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and S. W. Hell, “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

G. Moneron and S. W. Hell, “Two-photon excitation STED microscopy,” Opt. Express 17(17), 14567–14573 (2009).
[CrossRef] [PubMed]

U. V. Nägerl, K. I. Willig, B. Hein, S. W. Hell, and T. Bonhoeffer, “Live-cell imaging of dendritic spines by STED microscopy,” Proc. Natl. Acad. Sci. U.S.A. 105(48), 18982–18987 (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]

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[CrossRef] [PubMed]

E. Rittweger, B. R. Rankin, V. Westphal, and S. W. Hell, “Fluorescence depletion mechanisms in super-resolving STED microscopy,” Chem. Phys. Lett. 442(4-6), 483–487 (2007).
[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]

S. W. Hell, “Increasing the resolution of far-field fluorescence light microscopy by point-spread-function engineering,” Top. Fluoresc. Spectrosc. 5, 361–426 (2002).
[CrossRef]

M. Dyba, T. A. Klar, S. Jakobs, and S. W. Hell, “Ultrafast dynamics microscopy,” Appl. Phys. Lett. 77(4), 597–599 (2000).
[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]

T. A. Klar and S. W. Hell, “Subdiffraction resolution in far-field fluorescence microscopy,” Opt. Lett. 24(14), 954–956 (1999).
[CrossRef] [PubMed]

S. W. Hell and M. Kroug, “Ground-state-depletion fluorscence microscopy: A concept for breaking the diffraction resolution limit,” Appl. Phys. B 60(5), 495–497 (1995).
[CrossRef]

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

Hulit, J.

K. D. Piatkevich, J. Hulit, O. M. Subach, B. Wu, A. Abdulla, J. E. Segall, and V. V. Verkhusha, “Monomeric red fluorescent proteins with a large Stokes shift,” Proc. Natl. Acad. Sci. U.S.A. 107(12), 5369–5374 (2010).
[CrossRef] [PubMed]

Jakobs, S.

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and S. W. Hell, “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

M. Dyba, T. A. Klar, S. Jakobs, and S. W. Hell, “Ultrafast dynamics microscopy,” Appl. Phys. Lett. 77(4), 597–599 (2000).
[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]

Jovin, T. M.

Kastrup, L.

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]

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]

M. Dyba, T. A. Klar, S. Jakobs, and S. W. Hell, “Ultrafast dynamics microscopy,” Appl. Phys. Lett. 77(4), 597–599 (2000).
[CrossRef]

T. A. Klar and S. W. Hell, “Subdiffraction resolution in far-field fluorescence microscopy,” Opt. Lett. 24(14), 954–956 (1999).
[CrossRef] [PubMed]

Kroug, M.

S. W. Hell and M. Kroug, “Ground-state-depletion fluorscence microscopy: A concept for breaking the diffraction resolution limit,” Appl. Phys. B 60(5), 495–497 (1995).
[CrossRef]

Kusba, J.

I. Gryczynski, J. Kuśba, V. Bogdanov, and J. R. Lakowicz, “Quenching of fluorescence by light: A new method to control the excited-state lifetimes and orientations of fluorophores,” J. Fluoresc. 4(1), 103–109 (1994).
[CrossRef]

J. Kuśba, V. Bogdanov, I. Gryczynski, and J. R. Lakowicz, “Theory of light quenching: effects of fluorescence polarization, intensity, and anisotropy decays,” Biophys. J. 67(5), 2024–2040 (1994).
[CrossRef] [PubMed]

Lakowicz, J. R.

J. Kuśba, V. Bogdanov, I. Gryczynski, and J. R. Lakowicz, “Theory of light quenching: effects of fluorescence polarization, intensity, and anisotropy decays,” Biophys. J. 67(5), 2024–2040 (1994).
[CrossRef] [PubMed]

I. Gryczynski, J. Kuśba, V. Bogdanov, and J. R. Lakowicz, “Quenching of fluorescence by light: A new method to control the excited-state lifetimes and orientations of fluorophores,” J. Fluoresc. 4(1), 103–109 (1994).
[CrossRef]

I. Gryczynski, V. Bogdanov, and J. R. Lakowicz, “Light quenching of tetraphenylbutadiene fluorescence observed during two-photon excitation,” J. Fluoresc. 3(2), 85–92 (1993).
[CrossRef]

Lanigan, P. M. P.

Lesiecki, M.

M. Lesiecki, F. Asmar, J. M. Drake, and D. M. Camaioni, “Photoproperties of DCM,” J. Lumin. 31–32, 546–548 (1984).
[CrossRef]

Lesiecki, M. L.

J. M. Drake, M. L. Lesiecki, and D. M. Camaioni, “Photophysics and cis-trans isomerization of DCM,” Chem. Phys. Lett. 113(6), 530–534 (1985).
[CrossRef]

Li, Q.

Q. Li, S. S. Wu, and K. C. Chou, “Subdiffraction-limit two-photon fluorescence microscopy for GFP-tagged cell imaging,” Biophys. J. 97(12), 3224–3228 (2009).
[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]

Marsh, R. J.

A. J. Bain, R. J. Marsh, D. A. Armoogum, O. Mongin, L. Porrès, and M. Blanchard-Desce, “Time-resolved stimulated emission depletion in two-photon excited states,” Biochem. Soc. Trans. 31(5), 1047–1051 (2003).
[CrossRef] [PubMed]

R. J. Marsh, D. A. Armoogum, and A. J. Bain, “Stimulated emission depletion of two-photon excited states,” Chem. Phys. Lett. 366(3-4), 398–405 (2002).
[CrossRef]

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(11), 4258–4272 (2006).
[CrossRef] [PubMed]

Medda, R.

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[CrossRef] [PubMed]

Moneron, G.

Mongin, O.

A. J. Bain, R. J. Marsh, D. A. Armoogum, O. Mongin, L. Porrès, and M. Blanchard-Desce, “Time-resolved stimulated emission depletion in two-photon excited states,” Biochem. Soc. Trans. 31(5), 1047–1051 (2003).
[CrossRef] [PubMed]

Nägerl, U. V.

U. V. Nägerl, K. I. Willig, B. Hein, S. W. Hell, and T. Bonhoeffer, “Live-cell imaging of dendritic spines by STED microscopy,” Proc. Natl. Acad. Sci. U.S.A. 105(48), 18982–18987 (2008).
[CrossRef] [PubMed]

Neil, M. A. A.

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]

Piatkevich, K. D.

K. D. Piatkevich, J. Hulit, O. M. Subach, B. Wu, A. Abdulla, J. E. Segall, and V. V. Verkhusha, “Monomeric red fluorescent proteins with a large Stokes shift,” Proc. Natl. Acad. Sci. U.S.A. 107(12), 5369–5374 (2010).
[CrossRef] [PubMed]

Porrès, L.

A. J. Bain, R. J. Marsh, D. A. Armoogum, O. Mongin, L. Porrès, and M. Blanchard-Desce, “Time-resolved stimulated emission depletion in two-photon excited states,” Biochem. Soc. Trans. 31(5), 1047–1051 (2003).
[CrossRef] [PubMed]

Rankin, B. R.

E. Rittweger, B. R. Rankin, V. Westphal, and S. W. Hell, “Fluorescence depletion mechanisms in super-resolving STED microscopy,” Chem. Phys. Lett. 442(4-6), 483–487 (2007).
[CrossRef]

Rittweger, E.

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]

E. Rittweger, B. R. Rankin, V. Westphal, and S. W. Hell, “Fluorescence depletion mechanisms in super-resolving STED microscopy,” Chem. Phys. Lett. 442(4-6), 483–487 (2007).
[CrossRef]

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]

Sabatini, B. L.

J. B. Ding, K. T. Takasaki, and B. L. Sabatini, “Supraresolution imaging in brain slices using stimulated-emission depletion two-photon laser scanning microscopy,” Neuron 63(4), 429–437 (2009).
[CrossRef] [PubMed]

Segall, J. E.

K. D. Piatkevich, J. Hulit, O. M. Subach, B. Wu, A. Abdulla, J. E. Segall, and V. V. Verkhusha, “Monomeric red fluorescent proteins with a large Stokes shift,” Proc. Natl. Acad. Sci. U.S.A. 107(12), 5369–5374 (2010).
[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]

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Subach, O. M.

K. D. Piatkevich, J. Hulit, O. M. Subach, B. Wu, A. Abdulla, J. E. Segall, and V. V. Verkhusha, “Monomeric red fluorescent proteins with a large Stokes shift,” Proc. Natl. Acad. Sci. U.S.A. 107(12), 5369–5374 (2010).
[CrossRef] [PubMed]

Takasaki, K. T.

J. B. Ding, K. T. Takasaki, and B. L. Sabatini, “Supraresolution imaging in brain slices using stimulated-emission depletion two-photon laser scanning microscopy,” Neuron 63(4), 429–437 (2009).
[CrossRef] [PubMed]

Verkhusha, V. V.

K. D. Piatkevich, J. Hulit, O. M. Subach, B. Wu, A. Abdulla, J. E. Segall, and V. V. Verkhusha, “Monomeric red fluorescent proteins with a large Stokes shift,” Proc. Natl. Acad. Sci. U.S.A. 107(12), 5369–5374 (2010).
[CrossRef] [PubMed]

Webb, W. W.

Westphal, V.

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and S. W. Hell, “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

E. Rittweger, B. R. Rankin, V. Westphal, and S. W. Hell, “Fluorescence depletion mechanisms in super-resolving STED microscopy,” Chem. Phys. Lett. 442(4-6), 483–487 (2007).
[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]

Wichmann, J.

Wildanger, D.

Willig, K. I.

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and S. W. Hell, “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

U. V. Nägerl, K. I. Willig, B. Hein, S. W. Hell, and T. Bonhoeffer, “Live-cell imaging of dendritic spines by STED microscopy,” Proc. Natl. Acad. Sci. U.S.A. 105(48), 18982–18987 (2008).
[CrossRef] [PubMed]

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[CrossRef] [PubMed]

Wu, B.

K. D. Piatkevich, J. Hulit, O. M. Subach, B. Wu, A. Abdulla, J. E. Segall, and V. V. Verkhusha, “Monomeric red fluorescent proteins with a large Stokes shift,” Proc. Natl. Acad. Sci. U.S.A. 107(12), 5369–5374 (2010).
[CrossRef] [PubMed]

Wu, S. S.

Q. Li, S. S. Wu, and K. C. Chou, “Subdiffraction-limit two-photon fluorescence microscopy for GFP-tagged cell imaging,” Biophys. J. 97(12), 3224–3228 (2009).
[CrossRef] [PubMed]

Wurm, C. A.

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and S. W. Hell, “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

Xia, A.

X. Guo and A. Xia, “Ultrafast excited states relaxation dynamics in solution investigated by stimulated emission from a styryl dye,” J. Lumin. 122–123, 532–535 (2007).
[CrossRef]

Xu, C.

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]

Appl. Phys. B (1)

S. W. Hell and M. Kroug, “Ground-state-depletion fluorscence microscopy: A concept for breaking the diffraction resolution limit,” Appl. Phys. B 60(5), 495–497 (1995).
[CrossRef]

Appl. Phys. Lett. (2)

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]

M. Dyba, T. A. Klar, S. Jakobs, and S. W. Hell, “Ultrafast dynamics microscopy,” Appl. Phys. Lett. 77(4), 597–599 (2000).
[CrossRef]

Biochem. Soc. Trans. (1)

A. J. Bain, R. J. Marsh, D. A. Armoogum, O. Mongin, L. Porrès, and M. Blanchard-Desce, “Time-resolved stimulated emission depletion in two-photon excited states,” Biochem. Soc. Trans. 31(5), 1047–1051 (2003).
[CrossRef] [PubMed]

Biophys. J. (4)

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

B. Hein, K. I. Willig, C. A. Wurm, V. Westphal, S. Jakobs, and S. W. Hell, “Stimulated emission depletion nanoscopy of living cells using SNAP-tag fusion proteins,” Biophys. J. 98(1), 158–163 (2010).
[CrossRef] [PubMed]

Q. Li, S. S. Wu, and K. C. Chou, “Subdiffraction-limit two-photon fluorescence microscopy for GFP-tagged cell imaging,” Biophys. J. 97(12), 3224–3228 (2009).
[CrossRef] [PubMed]

J. Kuśba, V. Bogdanov, I. Gryczynski, and J. R. Lakowicz, “Theory of light quenching: effects of fluorescence polarization, intensity, and anisotropy decays,” Biophys. J. 67(5), 2024–2040 (1994).
[CrossRef] [PubMed]

Chem. Phys. Lett. (3)

R. J. Marsh, D. A. Armoogum, and A. J. Bain, “Stimulated emission depletion of two-photon excited states,” Chem. Phys. Lett. 366(3-4), 398–405 (2002).
[CrossRef]

J. M. Drake, M. L. Lesiecki, and D. M. Camaioni, “Photophysics and cis-trans isomerization of DCM,” Chem. Phys. Lett. 113(6), 530–534 (1985).
[CrossRef]

E. Rittweger, B. R. Rankin, V. Westphal, and S. W. Hell, “Fluorescence depletion mechanisms in super-resolving STED microscopy,” Chem. Phys. Lett. 442(4-6), 483–487 (2007).
[CrossRef]

J. Fluoresc. (2)

I. Gryczynski, V. Bogdanov, and J. R. Lakowicz, “Light quenching of tetraphenylbutadiene fluorescence observed during two-photon excitation,” J. Fluoresc. 3(2), 85–92 (1993).
[CrossRef]

I. Gryczynski, J. Kuśba, V. Bogdanov, and J. R. Lakowicz, “Quenching of fluorescence by light: A new method to control the excited-state lifetimes and orientations of fluorophores,” J. Fluoresc. 4(1), 103–109 (1994).
[CrossRef]

J. Lumin. (2)

M. Lesiecki, F. Asmar, J. M. Drake, and D. M. Camaioni, “Photoproperties of DCM,” J. Lumin. 31–32, 546–548 (1984).
[CrossRef]

X. Guo and A. Xia, “Ultrafast excited states relaxation dynamics in solution investigated by stimulated emission from a styryl dye,” J. Lumin. 122–123, 532–535 (2007).
[CrossRef]

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

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

Nat. Methods (2)

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[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]

Neuron (1)

J. B. Ding, K. T. Takasaki, and B. L. Sabatini, “Supraresolution imaging in brain slices using stimulated-emission depletion two-photon laser scanning microscopy,” Neuron 63(4), 429–437 (2009).
[CrossRef] [PubMed]

Opt. Commun. (1)

P. R. Hammond, “Laser dye DCM, its spectral properties, synthesis and comparison with other dyes in the red,” Opt. Commun. 29(3), 331–333 (1979).
[CrossRef]

Opt. Express (2)

Opt. Lett. (3)

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

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]

K. D. Piatkevich, J. Hulit, O. M. Subach, B. Wu, A. Abdulla, J. E. Segall, and V. V. Verkhusha, “Monomeric red fluorescent proteins with a large Stokes shift,” Proc. Natl. Acad. Sci. U.S.A. 107(12), 5369–5374 (2010).
[CrossRef] [PubMed]

U. V. Nägerl, K. I. Willig, B. Hein, S. W. Hell, and T. Bonhoeffer, “Live-cell imaging of dendritic spines by STED microscopy,” Proc. Natl. Acad. Sci. U.S.A. 105(48), 18982–18987 (2008).
[CrossRef] [PubMed]

M. G. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A. 102(37), 13081–13086 (2005).
[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]

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[CrossRef] [PubMed]

Top. Fluoresc. Spectrosc. (1)

S. W. Hell, “Increasing the resolution of far-field fluorescence light microscopy by point-spread-function engineering,” Top. Fluoresc. Spectrosc. 5, 361–426 (2002).
[CrossRef]

Other (1)

S. C. Baer, “Single wavelength stimulated emission depletion microscopy,” U.S. Patent 7,816,654 B2 (Oct. 19, 2010).

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Figures (9)

Fig. 1
Fig. 1

Energy levels and optical transitions involved in SW-STED.

Fig. 2
Fig. 2

The experimental setup.

Fig. 3
Fig. 3

DCM two-photon absorption cross section (straight line and squares) and fluorescence (dotted line) spectra in DMSO. The arrow indicates the excitation and stimulation wavelength. DCM chemical structure is shown in the inset.

Fig. 4
Fig. 4

Images and time evolutions of fluorescence emission in the DCM cell. Upper part: images taken with a CCD camera on the side of the sample cell inside which either the excitation beam (fs pulse) alone (a), or the STED beam (ps pulse) alone (b) are focused, or the two beams are overlapped (c). Lower part: fluorescence decays recorded with TCSPC device: (d), (e) and (f) correspond to images (a), (b) and (c) respectively. Average powers: 25 mW for excitation beam, 80 mW for STED beam.

Fig. 5
Fig. 5

Two-photon excited fluorescence quenched by a STED pulse delayed of 0.5 ns: (I) fluorescence decay without STED pulse; (II) fluorescence decay in presence of the STED pulse.

Fig. 6
Fig. 6

Depletion efficiency for excitation and STED beams with crossed polarization (a) and circular polarization (b). In both graphs, (I) is the undepleted decay (no STED beam) and (II) is the fluorescence decay in presence of the STED beam.

Fig. 7
Fig. 7

Fluorescence depletion as function of the peak intensity of the focused STED beam for excitation/stimulation at 680 nm (filled squares) and 700 nm (circles).

Fig. 8
Fig. 8

Calculated fluorescence depletion as a function of STED beam intensity in the single wavelength scheme (solid line). The beginning of the curve was fitted by an exponential decay (dashed line).

Fig. 9
Fig. 9

Calculated point spread function (PSF) and optical transfer function (OTF) for a SW-STED microscope with increasing peak intensity in the STED beam (the case ISTED = 0 corresponds to a conventional two-photon microscope with a pinhole). The excitation and STED beam profiles are shown in the inset. The calculations are based on a NA = 1.4 microscope objective, a laser wavelength of 680nm and a configuration with a pinhole in the image plane.

Equations (7)

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

d n 1 v i b d t = σ T P A ( I f e m t o 2 + I p i c o 2 ) ( n 0 n 1 v i b ) n 1 v i b τ v i b
d n 1 d t = σ S T E D I p i c o ( n 0 v i b n 1 ) n 1 ( 1 τ r a d + 1 τ N R ) + n 1 v i b τ v i b
d n 0 v i b d t = σ S T E D I p i c o ( n 1 n 0 v i b ) + n 1 ( 1 τ r a d + 1 τ N R ) n 0 v i b τ v i b
d n 0 d t = σ T P A ( I f e m t o 2 + I p i c o 2 ) ( n 0 n 1 v i b ) + n 0 v i b τ v i b
d n 1 d t = σ S T E D I p i c o n 1
n 1 e σ S T E D I P i c o Δ t
σ S T E D = 7. 10 18 c m 2 @ 680 n m σ S T E D = 3. 10 18 c m 2 @ 700 n m

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