Abstract

In stimulated emission depletion (STED) microscopy, the spatial resolution scales as the inverse square root of the STED beam's intensity. However, to fully exploit the maximum effective resolution achievable for a given STED beam’s intensity, several experimental precautions have to be considered. We focus our attention on the temporal alignment between the excitation and STED pulses and the polarization state of the STED beam. We present a simple theoretical framework that help to explain their influence on the performance of a STED microscope and we validate the results by imaging calibration and biological samples with a custom made STED architecture based on a supercontinuum laser source. We also highlight the advantages of using time gating detection in terms of temporal alignment.

© 2012 OSA

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    [Crossref] [PubMed]
  26. 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]
  27. 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]
  28. X. Hao, C. Kuang, T. Wang, and X. Liu, “Effects of polarization of the de-excitation dark focal spot in STED microscopy,” J. Opt. 12(11), 115707 (2010).
    [Crossref]
  29. P. Dedecker, B. Muls, J. Hofkens, J. Enderlein, and J.-I. Hotta, “Orientational effects in the excitation and de-excitation of single molecules interacting with donut-mode laser beams,” Opt. Express 15(6), 3372–3383 (2007).
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  31. S. Deng, L. Liu, Y. Cheng, R. Li, and Z. Xu, “Effects of primary aberrations on the fluorescence depletion patterns of STED microscopy,” Opt. Express 18(2), 1657–1666 (2010).
    [Crossref] [PubMed]
  32. J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express 15(6), 3361–3371 (2007).
    [Crossref] [PubMed]
  33. L. Qu, Y. Akbergenova, Y. Hu, and T. Schikorski, “Synapse-to-synapse variation in mean synaptic vesicle size and its relationship with synaptic morphology and function,” J. Comp. Neurol. 514(4), 343–352 (2009).
    [Crossref] [PubMed]
  34. 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]
  35. B. R. Rankin, R. R. Kellner, and S. W. Hell, “Stimulated-emission-depletion microscopy with a multicolor stimulated-Raman-scattering light source,” Opt. Lett. 33(21), 2491–2493 (2008).
    [Crossref] [PubMed]

2011 (3)

2010 (4)

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]

X. Hao, C. Kuang, T. Wang, and X. Liu, “Effects of polarization of the de-excitation dark focal spot in STED microscopy,” J. Opt. 12(11), 115707 (2010).
[Crossref]

S. Deng, L. Liu, Y. Cheng, R. Li, and Z. Xu, “Effects of primary aberrations on the fluorescence depletion patterns of STED microscopy,” Opt. Express 18(2), 1657–1666 (2010).
[Crossref] [PubMed]

2009 (8)

L. Qu, Y. Akbergenova, Y. Hu, and T. Schikorski, “Synapse-to-synapse variation in mean synaptic vesicle size and its relationship with synaptic morphology and function,” J. Comp. Neurol. 514(4), 343–352 (2009).
[Crossref] [PubMed]

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]

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]

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]

B. R. Rankin and S. W. Hell, “STED microscopy with a MHz pulsed stimulated-Raman-scattering source,” Opt. Express 17(18), 15679–15684 (2009).
[Crossref] [PubMed]

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

2008 (8)

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

B. Harke, C. K. Ullal, J. Keller, and S. W. Hell, “Three-dimensional nanoscopy of colloidal crystals,” Nano Lett. 8(5), 1309–1313 (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]

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. R. Rankin, R. R. Kellner, and S. W. Hell, “Stimulated-emission-depletion microscopy with a multicolor stimulated-Raman-scattering light source,” Opt. Lett. 33(21), 2491–2493 (2008).
[Crossref] [PubMed]

C.-K. Chou, W.-L. Chen, P. T. Fwu, S.-J. Lin, H.-S. Lee, and C.-Y. Dong, “Polarization ellipticity compensation in polarization second-harmonic generation microscopy without specimen rotation,” J. Biomed. Opt. 13(1), 014005 (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]

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]

2007 (4)

2006 (1)

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]

2003 (1)

1986 (1)

F. M. Wurm, K. A. Gwinn, and R. E. Kingston, “Inducible overproduction of the mouse c-myc protein in mammalian cells,” Proc. Natl. Acad. Sci. U.S.A. 83(15), 5414–5418 (1986).
[Crossref] [PubMed]

1873 (1)

E. Abbe, “Beitrage zur theorie des mikroskops und der mikroskopischen wahrnehmung,” Arch. Mikrosk. Anat. 9(1), 413–418 (1873).
[Crossref]

Abbe, E.

E. Abbe, “Beitrage zur theorie des mikroskops und der mikroskopischen wahrnehmung,” Arch. Mikrosk. Anat. 9(1), 413–418 (1873).
[Crossref]

Akbergenova, Y.

L. Qu, Y. Akbergenova, Y. Hu, and T. Schikorski, “Synapse-to-synapse variation in mean synaptic vesicle size and its relationship with synaptic morphology and function,” J. Comp. Neurol. 514(4), 343–352 (2009).
[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.

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.

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]

Boccacci, P.

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]

Boruah, B. R.

Bückers, J.

Chen, W.-L.

C.-K. Chou, W.-L. Chen, P. T. Fwu, S.-J. Lin, H.-S. Lee, and C.-Y. Dong, “Polarization ellipticity compensation in polarization second-harmonic generation microscopy without specimen rotation,” J. Biomed. Opt. 13(1), 014005 (2008).
[Crossref] [PubMed]

Cheng, Y.

Chou, C.-K.

C.-K. Chou, W.-L. Chen, P. T. Fwu, S.-J. Lin, H.-S. Lee, and C.-Y. Dong, “Polarization ellipticity compensation in polarization second-harmonic generation microscopy without specimen rotation,” J. Biomed. Opt. 13(1), 014005 (2008).
[Crossref] [PubMed]

Dedecker, P.

Deng, S.

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]

Dong, C.-Y.

C.-K. Chou, W.-L. Chen, P. T. Fwu, S.-J. Lin, H.-S. Lee, and C.-Y. Dong, “Polarization ellipticity compensation in polarization second-harmonic generation microscopy without specimen rotation,” J. Biomed. Opt. 13(1), 014005 (2008).
[Crossref] [PubMed]

Donnert, G.

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

Dunsby, C.

Dyba, M.

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]

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

Egner, A.

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]

Enderlein, J.

Engelhardt, J.

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]

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. W.

Fwu, P. T.

C.-K. Chou, W.-L. Chen, P. T. Fwu, S.-J. Lin, H.-S. Lee, and C.-Y. Dong, “Polarization ellipticity compensation in polarization second-harmonic generation microscopy without specimen rotation,” J. Biomed. Opt. 13(1), 014005 (2008).
[Crossref] [PubMed]

Gwinn, K. A.

F. M. Wurm, K. A. Gwinn, and R. E. Kingston, “Inducible overproduction of the mouse c-myc protein in mammalian cells,” Proc. Natl. Acad. Sci. U.S.A. 83(15), 5414–5418 (1986).
[Crossref] [PubMed]

Han, K. Y.

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]

Hao, X.

X. Hao, C. Kuang, T. Wang, and X. Liu, “Effects of polarization of the de-excitation dark focal spot in STED microscopy,” J. Opt. 12(11), 115707 (2010).
[Crossref]

Harke, B.

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]

B. Harke, C. K. Ullal, J. Keller, and S. W. Hell, “Three-dimensional nanoscopy of colloidal crystals,” Nano Lett. 8(5), 1309–1313 (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]

Hein, B.

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]

Hell, S. W.

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]

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

B. R. Rankin and S. W. Hell, “STED microscopy with a MHz pulsed stimulated-Raman-scattering source,” Opt. Express 17(18), 15679–15684 (2009).
[Crossref] [PubMed]

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

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

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. W. Hell, “Microscopy and its focal switch,” Nat. Methods 6(1), 24–32 (2009).
[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, C. K. Ullal, J. Keller, and S. W. Hell, “Three-dimensional nanoscopy of colloidal crystals,” Nano Lett. 8(5), 1309–1313 (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]

B. R. Rankin, R. R. Kellner, and S. W. Hell, “Stimulated-emission-depletion microscopy with a multicolor stimulated-Raman-scattering light source,” Opt. Lett. 33(21), 2491–2493 (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]

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]

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

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

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]

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

M. Dyba and S. W. Hell, “Photostability of a fluorescent marker under pulsed excited-state depletion through stimulated emission,” Appl. Opt. 42(25), 5123–5129 (2003).
[Crossref] [PubMed]

Hofkens, J.

Hotta, J.-I.

Hu, Y.

L. Qu, Y. Akbergenova, Y. Hu, and T. Schikorski, “Synapse-to-synapse variation in mean synaptic vesicle size and its relationship with synaptic morphology and function,” J. Comp. Neurol. 514(4), 343–352 (2009).
[Crossref] [PubMed]

Huang, B.

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

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.

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]

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]

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).
[Crossref] [PubMed]

B. Harke, C. K. Ullal, J. Keller, and S. W. Hell, “Three-dimensional nanoscopy of colloidal crystals,” Nano Lett. 8(5), 1309–1313 (2008).
[Crossref] [PubMed]

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

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

Kellner, R. R.

Kennedy, G.

Kingston, R. E.

F. M. Wurm, K. A. Gwinn, and R. E. Kingston, “Inducible overproduction of the mouse c-myc protein in mammalian cells,” Proc. Natl. Acad. Sci. U.S.A. 83(15), 5414–5418 (1986).
[Crossref] [PubMed]

Kuang, C.

X. Hao, C. Kuang, T. Wang, and X. Liu, “Effects of polarization of the de-excitation dark focal spot in STED microscopy,” J. Opt. 12(11), 115707 (2010).
[Crossref]

Lanigan, P. M. P.

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]

Lee, H.-S.

C.-K. Chou, W.-L. Chen, P. T. Fwu, S.-J. Lin, H.-S. Lee, and C.-Y. Dong, “Polarization ellipticity compensation in polarization second-harmonic generation microscopy without specimen rotation,” J. Biomed. Opt. 13(1), 014005 (2008).
[Crossref] [PubMed]

Leutenegger, M.

Li, R.

Lin, S.-J.

C.-K. Chou, W.-L. Chen, P. T. Fwu, S.-J. Lin, H.-S. Lee, and C.-Y. Dong, “Polarization ellipticity compensation in polarization second-harmonic generation microscopy without specimen rotation,” J. Biomed. Opt. 13(1), 014005 (2008).
[Crossref] [PubMed]

Liu, L.

Liu, X.

X. Hao, C. Kuang, T. Wang, and X. Liu, “Effects of polarization of the de-excitation dark focal spot in STED microscopy,” J. Opt. 12(11), 115707 (2010).
[Crossref]

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]

Medda, R.

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

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]

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]

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]

Michaelis, J.

Moffitt, J. R.

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).
[Crossref] [PubMed]

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

Muls, B.

Neil, M. A. A.

Osseforth, C.

Polyakova, S.

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]

Qu, L.

L. Qu, Y. Akbergenova, Y. Hu, and T. Schikorski, “Synapse-to-synapse variation in mean synaptic vesicle size and its relationship with synaptic morphology and function,” J. Comp. Neurol. 514(4), 343–352 (2009).
[Crossref] [PubMed]

Rankin, B. R.

Reuss, M.

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]

Ringemann, 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]

Rittweger, E.

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]

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]

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]

Sandhoff, K.

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]

Schikorski, T.

L. Qu, Y. Akbergenova, Y. Hu, and T. Schikorski, “Synapse-to-synapse variation in mean synaptic vesicle size and its relationship with synaptic morphology and function,” J. Comp. Neurol. 514(4), 343–352 (2009).
[Crossref] [PubMed]

Schmidt, R.

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.

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]

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

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]

Ullal, C. K.

B. Harke, C. K. Ullal, J. Keller, and S. W. Hell, “Three-dimensional nanoscopy of colloidal crystals,” Nano Lett. 8(5), 1309–1313 (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]

Vicidomini, G.

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, 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]

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]

Wang, T.

X. Hao, C. Kuang, T. Wang, and X. Liu, “Effects of polarization of the de-excitation dark focal spot in STED microscopy,” J. Opt. 12(11), 115707 (2010).
[Crossref]

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]

Wildanger, D.

Willig, K. I.

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]

Wurm, C. A.

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]

Wurm, F. M.

F. M. Wurm, K. A. Gwinn, and R. E. Kingston, “Inducible overproduction of the mouse c-myc protein in mammalian cells,” Proc. Natl. Acad. Sci. U.S.A. 83(15), 5414–5418 (1986).
[Crossref] [PubMed]

Xu, Z.

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. Opt. (1)

Arch. Mikrosk. Anat. (1)

E. Abbe, “Beitrage zur theorie des mikroskops und der mikroskopischen wahrnehmung,” Arch. Mikrosk. Anat. 9(1), 413–418 (1873).
[Crossref]

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]

J. Biomed. Opt. (1)

C.-K. Chou, W.-L. Chen, P. T. Fwu, S.-J. Lin, H.-S. Lee, and C.-Y. Dong, “Polarization ellipticity compensation in polarization second-harmonic generation microscopy without specimen rotation,” J. Biomed. Opt. 13(1), 014005 (2008).
[Crossref] [PubMed]

J. Comp. Neurol. (1)

L. Qu, Y. Akbergenova, Y. Hu, and T. Schikorski, “Synapse-to-synapse variation in mean synaptic vesicle size and its relationship with synaptic morphology and function,” J. Comp. Neurol. 514(4), 343–352 (2009).
[Crossref] [PubMed]

J. Microsc. (2)

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]

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

J. Opt. (1)

X. Hao, C. Kuang, T. Wang, and X. Liu, “Effects of polarization of the de-excitation dark focal spot in STED microscopy,” J. Opt. 12(11), 115707 (2010).
[Crossref]

Nano Lett. (1)

B. Harke, C. K. Ullal, J. Keller, and S. W. Hell, “Three-dimensional nanoscopy of colloidal crystals,” Nano Lett. 8(5), 1309–1313 (2008).
[Crossref] [PubMed]

Nat. Methods (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]

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]

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 (10)

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

B. R. Rankin and S. W. Hell, “STED microscopy with a MHz pulsed stimulated-Raman-scattering source,” Opt. Express 17(18), 15679–15684 (2009).
[Crossref] [PubMed]

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

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

S. Deng, L. Liu, Y. Cheng, R. Li, and Z. Xu, “Effects of primary aberrations on the fluorescence depletion patterns of STED microscopy,” Opt. Express 18(2), 1657–1666 (2010).
[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]

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]

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

Opt. Lett. (2)

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

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]

F. M. Wurm, K. A. Gwinn, and R. E. Kingston, “Inducible overproduction of the mouse c-myc protein in mammalian cells,” Proc. Natl. Acad. Sci. U.S.A. 83(15), 5414–5418 (1986).
[Crossref] [PubMed]

Science (2)

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

Other (4)

A. Diaspro, Confocal and Two-Photon Microscopy (Wiley-Liss, 2002).

A. Diaspro, Nanoscopy and multidimensional optical fluorescence microscopy (Chapman and Hall/CRC, 2010).

S. W. Hell, “Far-field optical nanoscopy,” in Single Molecule Spectroscopy in Chemistry, Physics and Biology, R. R. A. Graslund and J. Widengren, eds. (Springer, 2010), pp. 365–398.

S. W. Hell and A. Schonle, “Nanoscale resolution in far-field fluorescence microscopy,” in Science of Microscopy II, P. W. Hawkes and J. C. H. Spence, eds. (Springer, 2007), pp. 790–834.

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Figures (5)

Fig. 1
Fig. 1

Scheme of the STED architecture realized. BP: bandpass filter, DL: delay line, PBS: polarizing beam splitter, VPP: vortex phase plate, DM: dichroic mirror, OBJ: objective lens, S: sample, APD: avalanche photodiode.

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Fig. 2
Fig. 2

Calculated STED effective PSF for different incomplete inhibition factor δ (a), imperfections of the “zero”-intensity γ (b) and their combinations (c). Inset (a): fluorescence inhibition as function of the STED energy. Inset (b): radial distributions of intensity at the focus of the doughnut shape STED beam and Gaussian confocal beam. Calculations with dc = 200 nm, pd = 400 nm and ImSTEDTSTEDσSTED = 50.

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Fig. 3
Fig. 3

STED inhibition as function of the time delay between the excitation beam and the STED beams (a,b,c) (mean ± s.d; n = 10) . The inhibition is estimated respectively for the first STED beam acting solely (a), the second STED beam acting solely (b) and the two beams acting simultaneously (c). The yellow circles depict the position obtaining the highest depletion effect. In the case of time gated detection, inhibition is obtained by collecting the fluorescence immediately after the STED beams action. The inserted time-scale of the pulses are symbolic and not to scale. Comparison between STED and g-STED imaging of synaptic vesicles for different time delay between the excitation and the STED pulses (d). Excitation 572/15 nm, Detection 641/75 nm, Depletion 720/20 nm. Excitation average power ~4 µW (a-c) and ~0.6 µW (d); STED1 average power ~970 µW (a- c) and ~1.1 mW (d); STED2 average power ~970 µW. The scale bars in these images are 1 µm.

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Fig. 4
Fig. 4

(a) Remaining peak intensities between confocal and STED imaging for single isolated fluorescent beads as function of the STED beam polarization (mean ± s.e.m; n = 12). For each state of polarization we acquired three images: one in confocal modality, one in STED modality and the last again in confocal modality. We used the two confocal images to take into account the possible bleaching occurring during the imaging. (b) Comparison of synaptic vesicles imaging between confocal (upper panel) and STED with three different states of polarization. Excitation 572/15 nm, Detection 641/75 nm, Depletion 720/20 nm. Excitation average power ~4 µW (a) ~0.2 µW (b); STED average power ~970 µW (a) and ~1.1 mW .

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Fig. 5
Fig. 5

Resolution enhancement with STED microscopy. Fluorescent beads imaged in standard confocal mode (a), high-resolution STED (b) and the corresponding deconvolved ones (d, e). The plot (c) shows a confocal (grey), STED (red), confocal + deconvolution (thin black) and STED + deconvolution (thin dark red) line profiles along the arrows indicated in the images. Synaptic vesicles in GABAergic terminals of hippocampal neurons labeled with VGAT observed in standard confocal mode (f). In contrast, STED (g) reveals details of single vesicles which are unobservable in the confocal image. Both images show raw data. (i) and (j) show the corresponding deconvolved images. (h) Profile along the line indicated by arrows in the STED image reveals a resolution around 40 nm (red), corresponding to the average size of individual synaptic vesicles. For comparison we show also the corresponding profile of the deconvolved image (thin dark red). Excitation 572/15 nm, Detection 641/75 nm, Depletion 720/20 nm. Pixel size 15 nm. Excitation average power ~4 µW; STED average power ~4.2 mW. Scale bar of 0.5 µm. (d), (e), (i) and (j) are obtained after 10 iterations of a Richardoson-Lucy based deconvolution algorithm regularized by a moderate quadratic potential term.

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