Abstract

In stimulated emission depletion (STED) nanoscopy the wavelength of the STED beam is usually tuned towards the red tail of the emission maximum of the fluorophore. Shifting the STED wavelength closer to the emission peak, i.e. towards the blue region, favorably increases the stimulated emission cross-section. However, this blue-shifting also increases the probability to excite fluorophores that have remained in their ground state, compromising the image contrast. Here we present a method to exploit the higher STED efficiency of blue-shifted STED beams while maintaining the contrast in the image. The method is exemplified by imaging immunolabeled features in mammalian cells with an up to 3-fold increased STED efficiency compared to that encountered in standard STED nanoscopy implementations.

© 2012 OSA

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    [CrossRef] [PubMed]
  31. G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-color far-field fluorescence nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
    [CrossRef] [PubMed]
  32. 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]
  33. 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]
  34. P. A. Pellett, X. Sun, T. J. Gould, J. E. Rothman, M.-Q. Xu, I. R. Corrêa, and J. Bewersdorf, “Two-color STED microscopy in living cells,” Biomed. Opt. Express 2(8), 2364–2371 (2011).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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  39. G. Vicidomini, P. Boccacci, A. Diaspro, and M. Bertero, “Application of the split-gradient method to 3D image deconvolution in fluorescence microscopy,” J. Microsc. 234(1), 47–61 (2009).
    [CrossRef] [PubMed]

2011 (8)

B. R. Rankin, G. Moneron, C. A. Wurm, J. C. Nelson, A. Walter, D. Schwarzer, J. Schroeder, D. A. Colón-Ramos, and S. W. Hell, “Nanoscopy in a living multicellular organism expressing GFP,” Biophys. J. 100(12), L63–L65 (2011).
[CrossRef] [PubMed]

J. R. Moffitt, C. Osseforth, and J. Michaelis, “Time-gating improves the spatial resolution of STED microscopy,” Opt. Express 19(5), 4242–4254 (2011).
[CrossRef] [PubMed]

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[CrossRef] [PubMed]

T. Staudt, A. Engler, E. Rittweger, B. Harke, J. Engelhardt, and S. W. Hell, “Far-field optical nanoscopy with reduced number of state transition cycles,” Opt. Express 19(6), 5644–5657 (2011).
[CrossRef] [PubMed]

D. Aquino, A. Schönle, C. Geisler, C. V. Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, and A. Egner, “Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores,” Nat. Methods 8(4), 353–359 (2011).
[CrossRef] [PubMed]

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]

P. A. Pellett, X. Sun, T. J. Gould, J. E. Rothman, M.-Q. Xu, I. R. Corrêa, and J. Bewersdorf, “Two-color STED microscopy in living cells,” Biomed. Opt. Express 2(8), 2364–2371 (2011).
[CrossRef] [PubMed]

J. Tønnesen, F. Nadrigny, K. I. Willig, R. Wedlich-Söldner, and U. V. Nägerl, “Two-color STED microscopy of living synapses using a single laser-beam pair,” Biophys. J. 101(10), 2545–2552 (2011).
[CrossRef] [PubMed]

2010 (4)

G. Vicidomini, R. Schmidt, A. Egner, S. W. Hell, and A. Schönle, “Automatic deconvolution in 4Pi-microscopy with variable phase,” Opt. Express 18(10), 10154–10167 (2010).
[CrossRef] [PubMed]

J. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, “Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores,” J. Am. Chem. Soc. 132(14), 5021–5023 (2010).
[CrossRef] [PubMed]

M. Leutenegger, C. Eggeling, and S. W. Hell, “Analytical description of STED microscopy performance,” Opt. Express 18(25), 26417–26429 (2010).
[CrossRef] [PubMed]

B. Huang, H. Babcock, and X. Zhuang, “Breaking the diffraction barrier: super-resolution imaging of cells,” Cell 143(7), 1047–1058 (2010).
[CrossRef] [PubMed]

2009 (5)

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

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (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]

M. Bertero, P. Boccacci, G. Desiderà, and G. Vicidomini, “Image deblurring with poisson data: From cells to galaxies,” Inverse Probl. 25(12), 123006 (2009).
[CrossRef]

G. Vicidomini, P. Boccacci, A. Diaspro, and M. Bertero, “Application of the split-gradient method to 3D image deconvolution in fluorescence microscopy,” J. Microsc. 234(1), 47–61 (2009).
[CrossRef] [PubMed]

2008 (4)

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]

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

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]

2007 (5)

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

S. Bretschneider, C. Eggeling, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy by optical shelving,” Phys. Rev. Lett. 98(21), 218103 (2007).
[CrossRef] [PubMed]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-color far-field fluorescence nanoscopy,” Biophys. J. 92(8), L67–L69 (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]

2005 (1)

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]

2003 (1)

S. W. Hell, “Toward fluorescence nanoscopy,” Nat. Biotechnol. 21(11), 1347–1355 (2003).
[CrossRef] [PubMed]

2000 (1)

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]

1998 (1)

M. Yavuz and J. A. Fessler, “Statistical image reconstruction methods for randoms-precorrected PET scans,” Med. Image Anal. 2(4), 369–378 (1998).
[CrossRef] [PubMed]

1995 (1)

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

1994 (1)

1975 (1)

K. Weber, T. Bibring, and M. Osborn, “Specific visualization of tubulin-containing structures in tissue culture cells by immunofluorescence,” Exp. Cell Res. 95(1), 111–120 (1975).
[CrossRef] [PubMed]

1971 (1)

O. G. Peterson, J. P. Webb, W. C. McColgin, and J. H. Eberly, “Organic dye laser threshold,” J. Appl. Phys. 42(5), 1917–1928 (1971).
[CrossRef]

Aquino, D.

D. Aquino, A. Schönle, C. Geisler, C. V. Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, and A. Egner, “Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores,” Nat. Methods 8(4), 353–359 (2011).
[CrossRef] [PubMed]

Auksorius, E.

Babcock, H.

B. Huang, H. Babcock, and X. Zhuang, “Breaking the diffraction barrier: super-resolution imaging of cells,” Cell 143(7), 1047–1058 (2010).
[CrossRef] [PubMed]

Belov, V. N.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Bertero, M.

M. Bertero, P. Boccacci, G. Desiderà, and G. Vicidomini, “Image deblurring with poisson data: From cells to galaxies,” Inverse Probl. 25(12), 123006 (2009).
[CrossRef]

G. Vicidomini, P. Boccacci, A. Diaspro, and M. Bertero, “Application of the split-gradient method to 3D image deconvolution in fluorescence microscopy,” J. Microsc. 234(1), 47–61 (2009).
[CrossRef] [PubMed]

Bewersdorf, J.

Bibring, T.

K. Weber, T. Bibring, and M. Osborn, “Specific visualization of tubulin-containing structures in tissue culture cells by immunofluorescence,” Exp. Cell Res. 95(1), 111–120 (1975).
[CrossRef] [PubMed]

Boccacci, P.

M. Bertero, P. Boccacci, G. Desiderà, and G. Vicidomini, “Image deblurring with poisson data: From cells to galaxies,” Inverse Probl. 25(12), 123006 (2009).
[CrossRef]

G. Vicidomini, P. Boccacci, A. Diaspro, and M. Bertero, “Application of the split-gradient method to 3D image deconvolution in fluorescence microscopy,” J. Microsc. 234(1), 47–61 (2009).
[CrossRef] [PubMed]

Bock, H.

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature478, 204–208 (2011).

Boruah, B. R.

Bretschneider, S.

S. Bretschneider, C. Eggeling, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy by optical shelving,” Phys. Rev. Lett. 98(21), 218103 (2007).
[CrossRef] [PubMed]

Bückers, J.

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]

J. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, “Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores,” J. Am. Chem. Soc. 132(14), 5021–5023 (2010).
[CrossRef] [PubMed]

Colón-Ramos, D. A.

B. R. Rankin, G. Moneron, C. A. Wurm, J. C. Nelson, A. Walter, D. Schwarzer, J. Schroeder, D. A. Colón-Ramos, and S. W. Hell, “Nanoscopy in a living multicellular organism expressing GFP,” Biophys. J. 100(12), L63–L65 (2011).
[CrossRef] [PubMed]

Corrêa, I. R.

Dedecker, P.

J. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, “Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores,” J. Am. Chem. Soc. 132(14), 5021–5023 (2010).
[CrossRef] [PubMed]

Desiderà, G.

M. Bertero, P. Boccacci, G. Desiderà, and G. Vicidomini, “Image deblurring with poisson data: From cells to galaxies,” Inverse Probl. 25(12), 123006 (2009).
[CrossRef]

Diaspro, A.

G. Vicidomini, P. Boccacci, A. Diaspro, and M. Bertero, “Application of the split-gradient method to 3D image deconvolution in fluorescence microscopy,” J. Microsc. 234(1), 47–61 (2009).
[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]

Donnert, G.

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-color far-field fluorescence nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[CrossRef] [PubMed]

Dunsby, C.

Dyba, M.

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]

Eberly, J. H.

O. G. Peterson, J. P. Webb, W. C. McColgin, and J. H. Eberly, “Organic dye laser threshold,” J. Appl. Phys. 42(5), 1917–1928 (1971).
[CrossRef]

Eggeling, C.

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[CrossRef] [PubMed]

M. Leutenegger, C. Eggeling, and S. W. Hell, “Analytical description of STED microscopy performance,” Opt. Express 18(25), 26417–26429 (2010).
[CrossRef] [PubMed]

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

S. Bretschneider, C. Eggeling, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy by optical shelving,” Phys. Rev. Lett. 98(21), 218103 (2007).
[CrossRef] [PubMed]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-color far-field fluorescence nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[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]

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature478, 204–208 (2011).

Egner, A.

D. Aquino, A. Schönle, C. Geisler, C. V. Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, and A. Egner, “Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores,” Nat. Methods 8(4), 353–359 (2011).
[CrossRef] [PubMed]

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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).
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G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
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D. Aquino, A. Schönle, C. Geisler, C. V. Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, and A. Egner, “Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores,” Nat. Methods 8(4), 353–359 (2011).
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G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
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T. Staudt, A. Engler, E. Rittweger, B. Harke, J. Engelhardt, and S. W. Hell, “Far-field optical nanoscopy with reduced number of state transition cycles,” Opt. Express 19(6), 5644–5657 (2011).
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C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
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B. R. Rankin, G. Moneron, C. A. Wurm, J. C. Nelson, A. Walter, D. Schwarzer, J. Schroeder, D. A. Colón-Ramos, and S. W. Hell, “Nanoscopy in a living multicellular organism expressing GFP,” Biophys. J. 100(12), L63–L65 (2011).
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D. Aquino, A. Schönle, C. Geisler, C. V. Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, and A. Egner, “Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores,” Nat. Methods 8(4), 353–359 (2011).
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T. Staudt, A. Engler, E. Rittweger, B. Harke, J. Engelhardt, and S. W. Hell, “Far-field optical nanoscopy with reduced number of state transition cycles,” Opt. Express 19(6), 5644–5657 (2011).
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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).
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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).
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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).
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J. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, “Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores,” J. Am. Chem. Soc. 132(14), 5021–5023 (2010).
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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).
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G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-color far-field fluorescence nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
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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).
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J. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, “Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores,” J. Am. Chem. Soc. 132(14), 5021–5023 (2010).
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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).
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Kastrup, L.

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).
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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).
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S. W. Hell and M. Kroug, “Ground-state depletion fluorescence microscopy, a concept for breaking the diffraction resolution limit,” Appl. Phys. B 60(5), 495–497 (1995).
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D. Aquino, A. Schönle, C. Geisler, C. V. Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, and A. Egner, “Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores,” Nat. Methods 8(4), 353–359 (2011).
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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).
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T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature478, 204–208 (2011).

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M. Leutenegger, C. Eggeling, and S. W. Hell, “Analytical description of STED microscopy performance,” Opt. Express 18(25), 26417–26429 (2010).
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T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature478, 204–208 (2011).

Li, C.

J. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, “Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores,” J. Am. Chem. Soc. 132(14), 5021–5023 (2010).
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K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
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Middendorff, C. V.

D. Aquino, A. Schönle, C. Geisler, C. V. Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, and A. Egner, “Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores,” Nat. Methods 8(4), 353–359 (2011).
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Moneron, G.

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
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J. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, “Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores,” J. Am. Chem. Soc. 132(14), 5021–5023 (2010).
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Nelson, J. C.

B. R. Rankin, G. Moneron, C. A. Wurm, J. C. Nelson, A. Walter, D. Schwarzer, J. Schroeder, D. A. Colón-Ramos, and S. W. Hell, “Nanoscopy in a living multicellular organism expressing GFP,” Biophys. J. 100(12), L63–L65 (2011).
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D. Aquino, A. Schönle, C. Geisler, C. V. Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, and A. Egner, “Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores,” Nat. Methods 8(4), 353–359 (2011).
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C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
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B. R. Rankin, G. Moneron, C. A. Wurm, J. C. Nelson, A. Walter, D. Schwarzer, J. Schroeder, D. A. Colón-Ramos, and S. W. Hell, “Nanoscopy in a living multicellular organism expressing GFP,” Biophys. J. 100(12), L63–L65 (2011).
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G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
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C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
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T. Staudt, A. Engler, E. Rittweger, B. Harke, J. Engelhardt, and S. W. Hell, “Far-field optical nanoscopy with reduced number of state transition cycles,” Opt. Express 19(6), 5644–5657 (2011).
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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).
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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).
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G. Vicidomini, R. Schmidt, A. Egner, S. W. Hell, and A. Schönle, “Automatic deconvolution in 4Pi-microscopy with variable phase,” Opt. Express 18(10), 10154–10167 (2010).
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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).
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Schönle, A.

D. Aquino, A. Schönle, C. Geisler, C. V. Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, and A. Egner, “Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores,” Nat. Methods 8(4), 353–359 (2011).
[CrossRef] [PubMed]

G. Vicidomini, R. Schmidt, A. Egner, S. W. Hell, and A. Schönle, “Automatic deconvolution in 4Pi-microscopy with variable phase,” Opt. Express 18(10), 10154–10167 (2010).
[CrossRef] [PubMed]

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (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]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-color far-field fluorescence nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[CrossRef] [PubMed]

Schroeder, J.

B. R. Rankin, G. Moneron, C. A. Wurm, J. C. Nelson, A. Walter, D. Schwarzer, J. Schroeder, D. A. Colón-Ramos, and S. W. Hell, “Nanoscopy in a living multicellular organism expressing GFP,” Biophys. J. 100(12), L63–L65 (2011).
[CrossRef] [PubMed]

Schwarzer, D.

B. R. Rankin, G. Moneron, C. A. Wurm, J. C. Nelson, A. Walter, D. Schwarzer, J. Schroeder, D. A. Colón-Ramos, and S. W. Hell, “Nanoscopy in a living multicellular organism expressing GFP,” Biophys. J. 100(12), L63–L65 (2011).
[CrossRef] [PubMed]

Schwarzmann, G.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Staudt, T.

Sun, X.

Ta, H.

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[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]

Testa, I.

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature478, 204–208 (2011).

Tønnesen, J.

J. Tønnesen, F. Nadrigny, K. I. Willig, R. Wedlich-Söldner, and U. V. Nägerl, “Two-color STED microscopy of living synapses using a single laser-beam pair,” Biophys. J. 101(10), 2545–2552 (2011).
[CrossRef] [PubMed]

Ullal, C. K.

Urban, N. T.

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature478, 204–208 (2011).

Vicidomini, G.

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[CrossRef] [PubMed]

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]

G. Vicidomini, R. Schmidt, A. Egner, S. W. Hell, and A. Schönle, “Automatic deconvolution in 4Pi-microscopy with variable phase,” Opt. Express 18(10), 10154–10167 (2010).
[CrossRef] [PubMed]

M. Bertero, P. Boccacci, G. Desiderà, and G. Vicidomini, “Image deblurring with poisson data: From cells to galaxies,” Inverse Probl. 25(12), 123006 (2009).
[CrossRef]

G. Vicidomini, P. Boccacci, A. Diaspro, and M. Bertero, “Application of the split-gradient method to 3D image deconvolution in fluorescence microscopy,” J. Microsc. 234(1), 47–61 (2009).
[CrossRef] [PubMed]

von Middendorff, C.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Walter, A.

B. R. Rankin, G. Moneron, C. A. Wurm, J. C. Nelson, A. Walter, D. Schwarzer, J. Schroeder, D. A. Colón-Ramos, and S. W. Hell, “Nanoscopy in a living multicellular organism expressing GFP,” Biophys. J. 100(12), L63–L65 (2011).
[CrossRef] [PubMed]

Webb, J. P.

O. G. Peterson, J. P. Webb, W. C. McColgin, and J. H. Eberly, “Organic dye laser threshold,” J. Appl. Phys. 42(5), 1917–1928 (1971).
[CrossRef]

Weber, K.

K. Weber, T. Bibring, and M. Osborn, “Specific visualization of tubulin-containing structures in tissue culture cells by immunofluorescence,” Exp. Cell Res. 95(1), 111–120 (1975).
[CrossRef] [PubMed]

Wedlich-Söldner, R.

J. Tønnesen, F. Nadrigny, K. I. Willig, R. Wedlich-Söldner, and U. V. Nägerl, “Two-color STED microscopy of living synapses using a single laser-beam pair,” Biophys. J. 101(10), 2545–2552 (2011).
[CrossRef] [PubMed]

Westphal, V.

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[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]

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]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-color far-field fluorescence nanoscopy,” Biophys. J. 92(8), L67–L69 (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]

Wichmann, J.

Wildanger, D.

Willig, K. I.

J. Tønnesen, F. Nadrigny, K. I. Willig, R. Wedlich-Söldner, and U. V. Nägerl, “Two-color STED microscopy of living synapses using a single laser-beam pair,” Biophys. J. 101(10), 2545–2552 (2011).
[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]

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature478, 204–208 (2011).

Wurm, C. A.

B. R. Rankin, G. Moneron, C. A. Wurm, J. C. Nelson, A. Walter, D. Schwarzer, J. Schroeder, D. A. Colón-Ramos, and S. W. Hell, “Nanoscopy in a living multicellular organism expressing GFP,” Biophys. J. 100(12), L63–L65 (2011).
[CrossRef] [PubMed]

D. Aquino, A. Schönle, C. Geisler, C. V. Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, and A. Egner, “Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores,” Nat. Methods 8(4), 353–359 (2011).
[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]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-color far-field fluorescence nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[CrossRef] [PubMed]

Xu, M.-Q.

Yavuz, M.

M. Yavuz and J. A. Fessler, “Statistical image reconstruction methods for randoms-precorrected PET scans,” Med. Image Anal. 2(4), 369–378 (1998).
[CrossRef] [PubMed]

Zhuang, X.

B. Huang, H. Babcock, and X. Zhuang, “Breaking the diffraction barrier: super-resolution imaging of cells,” Cell 143(7), 1047–1058 (2010).
[CrossRef] [PubMed]

Appl. Phys. B (1)

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

Biomed. Opt. Express (1)

Biophys. J. (3)

J. Tønnesen, F. Nadrigny, K. I. Willig, R. Wedlich-Söldner, and U. V. Nägerl, “Two-color STED microscopy of living synapses using a single laser-beam pair,” Biophys. J. 101(10), 2545–2552 (2011).
[CrossRef] [PubMed]

G. Donnert, J. Keller, C. A. Wurm, S. O. Rizzoli, V. Westphal, A. Schönle, R. Jahn, S. Jakobs, C. Eggeling, and S. W. Hell, “Two-color far-field fluorescence nanoscopy,” Biophys. J. 92(8), L67–L69 (2007).
[CrossRef] [PubMed]

B. R. Rankin, G. Moneron, C. A. Wurm, J. C. Nelson, A. Walter, D. Schwarzer, J. Schroeder, D. A. Colón-Ramos, and S. W. Hell, “Nanoscopy in a living multicellular organism expressing GFP,” Biophys. J. 100(12), L63–L65 (2011).
[CrossRef] [PubMed]

Cell (1)

B. Huang, H. Babcock, and X. Zhuang, “Breaking the diffraction barrier: super-resolution imaging of cells,” Cell 143(7), 1047–1058 (2010).
[CrossRef] [PubMed]

Chem. Phys. Lett. (1)

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]

Exp. Cell Res. (1)

K. Weber, T. Bibring, and M. Osborn, “Specific visualization of tubulin-containing structures in tissue culture cells by immunofluorescence,” Exp. Cell Res. 95(1), 111–120 (1975).
[CrossRef] [PubMed]

Inverse Probl. (1)

M. Bertero, P. Boccacci, G. Desiderà, and G. Vicidomini, “Image deblurring with poisson data: From cells to galaxies,” Inverse Probl. 25(12), 123006 (2009).
[CrossRef]

J. Am. Chem. Soc. (1)

J. Hotta, E. Fron, P. Dedecker, K. P. F. Janssen, C. Li, K. Müllen, B. Harke, J. Bückers, S. W. Hell, and J. Hofkens, “Spectroscopic rationale for efficient stimulated-emission depletion microscopy fluorophores,” J. Am. Chem. Soc. 132(14), 5021–5023 (2010).
[CrossRef] [PubMed]

J. Appl. Phys. (1)

O. G. Peterson, J. P. Webb, W. C. McColgin, and J. H. Eberly, “Organic dye laser threshold,” J. Appl. Phys. 42(5), 1917–1928 (1971).
[CrossRef]

J. Microsc. (1)

G. Vicidomini, P. Boccacci, A. Diaspro, and M. Bertero, “Application of the split-gradient method to 3D image deconvolution in fluorescence microscopy,” J. Microsc. 234(1), 47–61 (2009).
[CrossRef] [PubMed]

Med. Image Anal. (1)

M. Yavuz and J. A. Fessler, “Statistical image reconstruction methods for randoms-precorrected PET scans,” Med. Image Anal. 2(4), 369–378 (1998).
[CrossRef] [PubMed]

Nat. Biotechnol. (1)

S. W. Hell, “Toward fluorescence nanoscopy,” Nat. Biotechnol. 21(11), 1347–1355 (2003).
[CrossRef] [PubMed]

Nat. Methods (5)

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]

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

D. Aquino, A. Schönle, C. Geisler, C. V. Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, and A. Egner, “Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores,” Nat. Methods 8(4), 353–359 (2011).
[CrossRef] [PubMed]

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[CrossRef] [PubMed]

Nature (1)

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[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. Express (6)

Opt. Lett. (2)

Phys. Rev. Lett. (1)

S. Bretschneider, C. Eggeling, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy by optical shelving,” Phys. Rev. Lett. 98(21), 218103 (2007).
[CrossRef] [PubMed]

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

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]

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]

Science (2)

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]

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

Other (5)

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature478, 204–208 (2011).

E. Abbe, Gesammelte abhandlungen (G. Fischer, Jena, 1904).

C. A. Wurm, D. Neumann, R. Schmidt, A. Egner, and S. Jakobs, “Sample preparation for STED microscopy live cell imaging,” D. B. Papkovsky, ed. (Humana Press, 2010), Chap. 11.

A. Giske, “CryoSTED microscopy: A new spectroscopic approach for improving the resolution of STED microscopy using low temperature,” PhD-Thesis (Ruperto-Carola University of Heidelberg, Heidelberg, 2007).

S. W. Hell and A. Schoenle, “Nanoscale resolution in far-field fluorescence microscopy,” in Science of microscopy, P. W. Hawkes and J. C. H. Spence, eds. (2007), Chap. 12.

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Figures (5)

Fig. 1
Fig. 1

Principle of the anti-Stokes excitation (AStEx) determination. (a,b) Experimental time sequence for (a) CW-STED and (b) P-STED implementations. The AStEx contribution is taken from the signal detected during Gate 2. Time-correlated single-photon counting histograms in log-scale of ATTO-647N labeled microtubules in PtK2 cells for (c) CW-STED and (d) P-STED configurations. For comparison, time-correlated single-photons histograms generated just by the excitation beam, i.e. without the STED beam, are also shown (gray lines).

Fig. 2
Fig. 2

Comparison of CW-STED nanoscopy operating at λSTED = 760 nm (upper panels) and 730 nm (lower panels): confocal (top left corners), raw gCW-STED (Gate1, left panels), anti Stokes excitation (AStEx) induced fluorescence generated by the STED beam (Gate 2, middle left panels) and recovered gCW-STED images (Gate1 – Gate2, middle right panels) of ATTO-647N labeled microtubules as well as normalized intensity profiles along the dashed lines (right panels). The asterisk in the color look-up table denotes that the negative counts obtained after subtraction are clipped to zero. Excitation: λex = 635nm, fex = 40 MHz and áPex ñ = 5 μW; STED: áPSTED ñ = 290 mW at λSTED = 760 nm and áPSTED ñ = 250 mW at 730 nm; time-gated detection: Tg = 1 ns and ΔT = 8 ns; Pixel size 20 nm; Pixel dwell-time 0.5 ms. In this and the following figures, the inserted focal excitation spots and doughnuts are symbolic and not to scale. Scale bars: 1μm.

Fig. 3
Fig. 3

Comparison of pulsed STED nanoscopy at λSTED = 760 nm (upper panels) and λSTED = 730 nm (lower panels). The figure depicts confocal (top left corners), raw gP-STED (Gate1, left panels), AStEx by the STED beam (Gate2, middle left panels) and recovered gP-STED images (Gate1- Gate2, middle right panels) of ATTO-647N labeled microtubules as well as normalized intensity profiles along the dashed lines (right panels). The asterisk denotes that negative counts following subtraction are clipped to zero. Excitation: λex = 635nm, fex = 76/2 MHz and áPex ñ = 5 μW; STED: fSTED = 76 MHz, áPSTED ñ = 70 mW at λSTED = 760 nm and áPSTED ñ = 60 mW at λSTED = 730 nm; time-gated detection: Tg = 1 ns and ΔT = 8 ns; Pixel size 20 nm; Pixel dwell-time 0.5 ms. Scale bars: 1μm.

Fig. 4
Fig. 4

Dependence of the FWHM determined from STED images of ATTO-647N-labeled microtubules on the average STED power for (a) the CW-STED and (b) the pulsed STED configuration (mean ± s.e.m.; n = 10). Solid lines show theoretical fits of Eq. (3) to the data with dc = 235 nm, β = 1.04·10−5 nm−2, τ = 3.15 ns, s = 50 nm, Tg = 1 ns and TSTED = 250 ps. The inset in (a) shows the absorption (blue) and emission (green) spectra of the fluorophore ATTO-647N and the applied STED wavelengths. Note that the time averaged power of the STED beam was measured before entering the lens. Due to losses in the lens, the power at the sample is actually lower by 30% and 25% at 760 nm and 730 nm, respectively.

Fig. 5
Fig. 5

Comparison of gP-STED nanoscopy imaging obtained with two spectrally different fluorophores, ATTO-565 (upper panels) and ATTO-Rho12 (lower panels), using the same excitation and STED beams. The figure depicts confocal (top left corners), raw gP-STED (Gate1, left panels), AStEx induced fluorescence from the STED beam (Gate2, middle panels) and recovered gP-STED images (Gate1 – Gate2, right panels) of labeled vimentin filaments. Excitation: λex = 532 nm, fex = 35.7 MHz and áPexñ = 1 µW; STED: λSTED = 647 nm, fSTED = 71.4 MHz, for ATTO Rho12 áPSTED ñ = 35 mW; for ATTO-565 áPSTED ñ = 78 mW time-gated detection: Tg = 0.1 ns and ΔT = 10 ns; Pixel size 19 nm; Pixel dwell-time 100 µs. Scale bars: 1μm.

Equations (3)

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

FWH M gP = d c / 1+ d c 2 β( I STED m / I s ) T STED / ( ln( 2 )τ )
FWH M gCW = d c / 1+ d c 2 β( I STED m / I s )( 1+ T g / ( ln( 2 )τ ) )
FWH M mt = FWH M gP / gCW 2 + s 2

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