Abstract

We present a novel concept adaptable to any kind of STED microscope in order to expand the limited number of compatible dyes for performing super resolution imaging. The approach is based on an intensity modulated excitation beam in combination with a frequency dependent detection in the form of a standard lock-in amplifier. This enables to unmix fluorescence signal originated by the excitation beam from the fluorescence caused by the STED beam. The benefit of this concept is demonstrated by imaging biological samples as well as fluorescent spheres, whose spectrum does not allow STED imaging in the conventional way. Our concept is suitable with CW or pulsed STED microscope and can thereby be seen as a general improvement adaptable to any existing setup.

© 2013 OSA

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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  9. B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express16(6), 4154–4162 (2008).
    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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  21. D. Wildanger, E. Rittweger, L. Kastrup, and S. W. Hell, “STED microscopy with a supercontinuum laser source,” Opt. Express16(13), 9614–9621 (2008).
    [CrossRef] [PubMed]
  22. 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,” Science320(5873), 246–249 (2008).
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  24. G. Vicidomini, G. Moneron, C. Eggeling, E. Rittweger, and S. W. Hell, “STED with wavelengths closer to the emission maximum,” Opt. Express20(5), 5225–5236 (2012).
    [CrossRef] [PubMed]
  25. R. Kasper, B. Harke, C. Forthmann, P. Tinnefeld, S. W. Hell, and M. Sauer, “Single-Molecule STED Microscopy with Photostable Organic Fluorophores,” Small 6, 1379–1384 (2010).
  26. 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. Express19(6), 5644–5657 (2011).
    [CrossRef] [PubMed]
  27. 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. Methods5(6), 539–544 (2008).
    [CrossRef] [PubMed]

2012

M. A. Lauterbach, “Finding, defining and breaking the diffraction barrier in microscopy - a historical perspective,” Optical Nanoscopy1(1), 8 (2012).
[CrossRef]

M. Leutenegger, C. Ringemann, T. Lasser, S. W. Hell, and C. Eggeling, “Fluorescence correlation spectroscopy with a total internal reflection fluorescence STED microscope (TIRF-STED-FCS),” Opt. Express20(5), 5243–5263 (2012).
[CrossRef] [PubMed]

P. Bianchini, B. Harke, S. Galiani, G. Vicidomini, and A. Diaspro, “Single-wavelength two-photon excitation-stimulated emission depletion (SW2PE-STED) superresolution imaging,” Proc. Natl. Acad. Sci. U.S.A.109(17), 6390–6393 (2012).
[CrossRef] [PubMed]

B. Harke, J. Varghese Chacko, C. Canale, H. Haschke, and A. Diaspro, “A novel nanoscopic tool by combining AFM with STED microscopy,” Optical Nanoscopy 1, (2012).

S. Galiani, B. Harke, G. Vicidomini, G. Lignani, F. Benfenati, A. Diaspro, and P. Bianchini, “Strategies to maximize the performance of a STED microscope,” Opt. Express20(7), 7362–7374 (2012).
[CrossRef] [PubMed]

G. Vicidomini, G. Moneron, C. Eggeling, E. Rittweger, and S. W. Hell, “STED with wavelengths closer to the emission maximum,” Opt. Express20(5), 5225–5236 (2012).
[CrossRef] [PubMed]

2011

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. Express19(6), 5644–5657 (2011).
[CrossRef] [PubMed]

T. J. Gould, J. R. Myers, and J. Bewersdorf, “Total internal reflection STED microscopy,” Opt. Express19(14), 13351–13357 (2011).
[CrossRef] [PubMed]

C. A. Wurm, D. Neumann, M. A. Lauterbach, B. Harke, A. Egner, S. W. Hell, and S. Jakobs, “Nanoscale distribution of mitochondrial import receptor Tom20 is adjusted to cellular conditions and exhibits an inner-cellular gradient,” Proc. Natl. Acad. Sci. U.S.A.108(33), 13546–13551 (2011).
[CrossRef] [PubMed]

2010

R. Kasper, B. Harke, C. Forthmann, P. Tinnefeld, S. W. Hell, and M. Sauer, “Single-Molecule STED Microscopy with Photostable Organic Fluorophores,” Small 6, 1379–1384 (2010).

2009

J. B. Ding, K. T. Takasaki, and B. L. Sabatini, “Supraresolution imaging in brain slices using stimulated-emission depletion two-photon laser scanning microscopy,” Neuron63(4), 429–437 (2009).
[CrossRef] [PubMed]

G. Moneron and S. W. Hell, “Two-photon excitation STED microscopy,” Opt. Express17(17), 14567–14573 (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,” Nature457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

S. W. Hell, “Microscopy and its focal switch,” Nat. Methods6(1), 24–32 (2009).
[CrossRef] [PubMed]

2008

2007

2006

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, “STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis,” Nature440(7086), 935–939 (2006).
[CrossRef] [PubMed]

2005

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

2003

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

1994

1873

E. Abbe, “Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung,” Archiv Für Mikroskopische Anatomie9(1), 413–418 (1873).
[CrossRef]

Abbe, E.

E. Abbe, “Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung,” Archiv Für Mikroskopische Anatomie9(1), 413–418 (1873).
[CrossRef]

Auksorius, E.

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,” Nature457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Benfenati, F.

Bewersdorf, J.

Bianchini, P.

P. Bianchini, B. Harke, S. Galiani, G. Vicidomini, and A. Diaspro, “Single-wavelength two-photon excitation-stimulated emission depletion (SW2PE-STED) superresolution imaging,” Proc. Natl. Acad. Sci. U.S.A.109(17), 6390–6393 (2012).
[CrossRef] [PubMed]

S. Galiani, B. Harke, G. Vicidomini, G. Lignani, F. Benfenati, A. Diaspro, and P. Bianchini, “Strategies to maximize the performance of a STED microscope,” Opt. Express20(7), 7362–7374 (2012).
[CrossRef] [PubMed]

Boruah, B. R.

Canale, C.

B. Harke, J. Varghese Chacko, C. Canale, H. Haschke, and A. Diaspro, “A novel nanoscopic tool by combining AFM with STED microscopy,” Optical Nanoscopy 1, (2012).

Chen, N.

Diaspro, A.

P. Bianchini, B. Harke, S. Galiani, G. Vicidomini, and A. Diaspro, “Single-wavelength two-photon excitation-stimulated emission depletion (SW2PE-STED) superresolution imaging,” Proc. Natl. Acad. Sci. U.S.A.109(17), 6390–6393 (2012).
[CrossRef] [PubMed]

S. Galiani, B. Harke, G. Vicidomini, G. Lignani, F. Benfenati, A. Diaspro, and P. Bianchini, “Strategies to maximize the performance of a STED microscope,” Opt. Express20(7), 7362–7374 (2012).
[CrossRef] [PubMed]

B. Harke, J. Varghese Chacko, C. Canale, H. Haschke, and A. Diaspro, “A novel nanoscopic tool by combining AFM with STED microscopy,” Optical Nanoscopy 1, (2012).

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,” Neuron63(4), 429–437 (2009).
[CrossRef] [PubMed]

Dunsby, C.

Eggeling, C.

Egner, A.

C. A. Wurm, D. Neumann, M. A. Lauterbach, B. Harke, A. Egner, S. W. Hell, and S. Jakobs, “Nanoscale distribution of mitochondrial import receptor Tom20 is adjusted to cellular conditions and exhibits an inner-cellular gradient,” Proc. Natl. Acad. Sci. U.S.A.108(33), 13546–13551 (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. Methods5(6), 539–544 (2008).
[CrossRef] [PubMed]

Engelhardt, J.

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. Express19(6), 5644–5657 (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. Methods5(6), 539–544 (2008).
[CrossRef] [PubMed]

Engler, A.

Forthmann, C.

R. Kasper, B. Harke, C. Forthmann, P. Tinnefeld, S. W. Hell, and M. Sauer, “Single-Molecule STED Microscopy with Photostable Organic Fluorophores,” Small 6, 1379–1384 (2010).

French, P. M. W.

Galiani, S.

S. Galiani, B. Harke, G. Vicidomini, G. Lignani, F. Benfenati, A. Diaspro, and P. Bianchini, “Strategies to maximize the performance of a STED microscope,” Opt. Express20(7), 7362–7374 (2012).
[CrossRef] [PubMed]

P. Bianchini, B. Harke, S. Galiani, G. Vicidomini, and A. Diaspro, “Single-wavelength two-photon excitation-stimulated emission depletion (SW2PE-STED) superresolution imaging,” Proc. Natl. Acad. Sci. U.S.A.109(17), 6390–6393 (2012).
[CrossRef] [PubMed]

Gould, T. J.

Harke, B.

P. Bianchini, B. Harke, S. Galiani, G. Vicidomini, and A. Diaspro, “Single-wavelength two-photon excitation-stimulated emission depletion (SW2PE-STED) superresolution imaging,” Proc. Natl. Acad. Sci. U.S.A.109(17), 6390–6393 (2012).
[CrossRef] [PubMed]

B. Harke, J. Varghese Chacko, C. Canale, H. Haschke, and A. Diaspro, “A novel nanoscopic tool by combining AFM with STED microscopy,” Optical Nanoscopy 1, (2012).

S. Galiani, B. Harke, G. Vicidomini, G. Lignani, F. Benfenati, A. Diaspro, and P. Bianchini, “Strategies to maximize the performance of a STED microscope,” Opt. Express20(7), 7362–7374 (2012).
[CrossRef] [PubMed]

C. A. Wurm, D. Neumann, M. A. Lauterbach, B. Harke, A. Egner, S. W. Hell, and S. Jakobs, “Nanoscale distribution of mitochondrial import receptor Tom20 is adjusted to cellular conditions and exhibits an inner-cellular gradient,” Proc. Natl. Acad. Sci. U.S.A.108(33), 13546–13551 (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. Express19(6), 5644–5657 (2011).
[CrossRef] [PubMed]

R. Kasper, B. Harke, C. Forthmann, P. Tinnefeld, S. W. Hell, and M. Sauer, “Single-Molecule STED Microscopy with Photostable Organic Fluorophores,” Small 6, 1379–1384 (2010).

B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express16(6), 4154–4162 (2008).
[CrossRef] [PubMed]

Haschke, H.

B. Harke, J. Varghese Chacko, C. Canale, H. Haschke, and A. Diaspro, “A novel nanoscopic tool by combining AFM with STED microscopy,” Optical Nanoscopy 1, (2012).

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,” Nature457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Hell, S. W.

M. Leutenegger, C. Ringemann, T. Lasser, S. W. Hell, and C. Eggeling, “Fluorescence correlation spectroscopy with a total internal reflection fluorescence STED microscope (TIRF-STED-FCS),” Opt. Express20(5), 5243–5263 (2012).
[CrossRef] [PubMed]

G. Vicidomini, G. Moneron, C. Eggeling, E. Rittweger, and S. W. Hell, “STED with wavelengths closer to the emission maximum,” Opt. Express20(5), 5225–5236 (2012).
[CrossRef] [PubMed]

C. A. Wurm, D. Neumann, M. A. Lauterbach, B. Harke, A. Egner, S. W. Hell, and S. Jakobs, “Nanoscale distribution of mitochondrial import receptor Tom20 is adjusted to cellular conditions and exhibits an inner-cellular gradient,” Proc. Natl. Acad. Sci. U.S.A.108(33), 13546–13551 (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. Express19(6), 5644–5657 (2011).
[CrossRef] [PubMed]

R. Kasper, B. Harke, C. Forthmann, P. Tinnefeld, S. W. Hell, and M. Sauer, “Single-Molecule STED Microscopy with Photostable Organic Fluorophores,” Small 6, 1379–1384 (2010).

G. Moneron and S. W. Hell, “Two-photon excitation STED microscopy,” Opt. Express17(17), 14567–14573 (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,” Nature457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

S. W. Hell, “Microscopy and its focal switch,” Nat. Methods6(1), 24–32 (2009).
[CrossRef] [PubMed]

B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express16(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,” Science320(5873), 246–249 (2008).
[CrossRef] [PubMed]

D. Wildanger, E. Rittweger, L. Kastrup, and S. W. Hell, “STED microscopy with a supercontinuum laser source,” Opt. Express16(13), 9614–9621 (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. Methods5(6), 539–544 (2008).
[CrossRef] [PubMed]

J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express15(6), 3361–3371 (2007).
[CrossRef] [PubMed]

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, “STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis,” Nature440(7086), 935–939 (2006).
[CrossRef] [PubMed]

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

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

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

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,” Science320(5873), 246–249 (2008).
[CrossRef] [PubMed]

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, “STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis,” Nature440(7086), 935–939 (2006).
[CrossRef] [PubMed]

Jakobs, S.

C. A. Wurm, D. Neumann, M. A. Lauterbach, B. Harke, A. Egner, S. W. Hell, and S. Jakobs, “Nanoscale distribution of mitochondrial import receptor Tom20 is adjusted to cellular conditions and exhibits an inner-cellular gradient,” Proc. Natl. Acad. Sci. U.S.A.108(33), 13546–13551 (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. Methods5(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,” Science320(5873), 246–249 (2008).
[CrossRef] [PubMed]

Kasper, R.

R. Kasper, B. Harke, C. Forthmann, P. Tinnefeld, S. W. Hell, and M. Sauer, “Single-Molecule STED Microscopy with Photostable Organic Fluorophores,” Small 6, 1379–1384 (2010).

Kastrup, L.

Keller, J.

Kennedy, G.

Lanigan, P. M. P.

Lasser, T.

Lauterbach, M. A.

M. A. Lauterbach, “Finding, defining and breaking the diffraction barrier in microscopy - a historical perspective,” Optical Nanoscopy1(1), 8 (2012).
[CrossRef]

C. A. Wurm, D. Neumann, M. A. Lauterbach, B. Harke, A. Egner, S. W. Hell, and S. Jakobs, “Nanoscale distribution of mitochondrial import receptor Tom20 is adjusted to cellular conditions and exhibits an inner-cellular gradient,” Proc. Natl. Acad. Sci. U.S.A.108(33), 13546–13551 (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,” Science320(5873), 246–249 (2008).
[CrossRef] [PubMed]

Leutenegger, M.

Lignani, G.

Medda, R.

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,” Nature457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Moneron, G.

Myers, J. R.

Neil, M. A. A.

Neumann, D.

C. A. Wurm, D. Neumann, M. A. Lauterbach, B. Harke, A. Egner, S. W. Hell, and S. Jakobs, “Nanoscale distribution of mitochondrial import receptor Tom20 is adjusted to cellular conditions and exhibits an inner-cellular gradient,” Proc. Natl. Acad. Sci. U.S.A.108(33), 13546–13551 (2011).
[CrossRef] [PubMed]

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,” Nature457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Ringemann, C.

M. Leutenegger, C. Ringemann, T. Lasser, S. W. Hell, and C. Eggeling, “Fluorescence correlation spectroscopy with a total internal reflection fluorescence STED microscope (TIRF-STED-FCS),” Opt. Express20(5), 5243–5263 (2012).
[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,” Nature457(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,” Science320(5873), 246–249 (2008).
[CrossRef] [PubMed]

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, “STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis,” Nature440(7086), 935–939 (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,” Neuron63(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,” Nature457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Sauer, M.

R. Kasper, B. Harke, C. Forthmann, P. Tinnefeld, S. W. Hell, and M. Sauer, “Single-Molecule STED Microscopy with Photostable Organic Fluorophores,” Small 6, 1379–1384 (2010).

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. Methods5(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,” Nature457(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. Express16(6), 4154–4162 (2008).
[CrossRef] [PubMed]

J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express15(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,” Nature457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Sheppard, C. J.

Staudt, T.

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,” Neuron63(4), 429–437 (2009).
[CrossRef] [PubMed]

Tinnefeld, P.

R. Kasper, B. Harke, C. Forthmann, P. Tinnefeld, S. W. Hell, and M. Sauer, “Single-Molecule STED Microscopy with Photostable Organic Fluorophores,” Small 6, 1379–1384 (2010).

Ullal, C. K.

Varghese Chacko, J.

B. Harke, J. Varghese Chacko, C. Canale, H. Haschke, and A. Diaspro, “A novel nanoscopic tool by combining AFM with STED microscopy,” Optical Nanoscopy 1, (2012).

Vicidomini, G.

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,” Nature457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Westphal, V.

B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express16(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,” Science320(5873), 246–249 (2008).
[CrossRef] [PubMed]

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, “STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis,” Nature440(7086), 935–939 (2006).
[CrossRef] [PubMed]

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

Wichmann, J.

Wildanger, D.

Willig, K. I.

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, “STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis,” Nature440(7086), 935–939 (2006).
[CrossRef] [PubMed]

Wong, C. H.

Wurm, C. A.

C. A. Wurm, D. Neumann, M. A. Lauterbach, B. Harke, A. Egner, S. W. Hell, and S. Jakobs, “Nanoscale distribution of mitochondrial import receptor Tom20 is adjusted to cellular conditions and exhibits an inner-cellular gradient,” Proc. Natl. Acad. Sci. U.S.A.108(33), 13546–13551 (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. Methods5(6), 539–544 (2008).
[CrossRef] [PubMed]

Archiv Für Mikroskopische Anatomie

E. Abbe, “Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung,” Archiv Für Mikroskopische Anatomie9(1), 413–418 (1873).
[CrossRef]

Nat. Biotechnol.

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

Nat. Methods

S. W. Hell, “Microscopy and its focal switch,” Nat. Methods6(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. Methods5(6), 539–544 (2008).
[CrossRef] [PubMed]

Nature

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, “STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis,” Nature440(7086), 935–939 (2006).
[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,” Nature457(7233), 1159–1162 (2009).
[CrossRef] [PubMed]

Neuron

J. B. Ding, K. T. Takasaki, and B. L. Sabatini, “Supraresolution imaging in brain slices using stimulated-emission depletion two-photon laser scanning microscopy,” Neuron63(4), 429–437 (2009).
[CrossRef] [PubMed]

Opt. Express

G. Moneron and S. W. Hell, “Two-photon excitation STED microscopy,” Opt. Express17(17), 14567–14573 (2009).
[CrossRef] [PubMed]

T. J. Gould, J. R. Myers, and J. Bewersdorf, “Total internal reflection STED microscopy,” Opt. Express19(14), 13351–13357 (2011).
[CrossRef] [PubMed]

M. Leutenegger, C. Ringemann, T. Lasser, S. W. Hell, and C. Eggeling, “Fluorescence correlation spectroscopy with a total internal reflection fluorescence STED microscope (TIRF-STED-FCS),” Opt. Express20(5), 5243–5263 (2012).
[CrossRef] [PubMed]

J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express15(6), 3361–3371 (2007).
[CrossRef] [PubMed]

B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express16(6), 4154–4162 (2008).
[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. Express19(6), 5644–5657 (2011).
[CrossRef] [PubMed]

N. Chen, C. H. Wong, and C. J. Sheppard, “Focal modulation microscopy,” Opt. Express16(23), 18764–18769 (2008).
[CrossRef] [PubMed]

G. Vicidomini, G. Moneron, C. Eggeling, E. Rittweger, and S. W. Hell, “STED with wavelengths closer to the emission maximum,” Opt. Express20(5), 5225–5236 (2012).
[CrossRef] [PubMed]

S. Galiani, B. Harke, G. Vicidomini, G. Lignani, F. Benfenati, A. Diaspro, and P. Bianchini, “Strategies to maximize the performance of a STED microscope,” Opt. Express20(7), 7362–7374 (2012).
[CrossRef] [PubMed]

D. Wildanger, E. Rittweger, L. Kastrup, and S. W. Hell, “STED microscopy with a supercontinuum laser source,” Opt. Express16(13), 9614–9621 (2008).
[CrossRef] [PubMed]

Opt. Lett.

Optical Nanoscopy

M. A. Lauterbach, “Finding, defining and breaking the diffraction barrier in microscopy - a historical perspective,” Optical Nanoscopy1(1), 8 (2012).
[CrossRef]

Phys. Rev. Lett.

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

Proc. Natl. Acad. Sci. U.S.A.

P. Bianchini, B. Harke, S. Galiani, G. Vicidomini, and A. Diaspro, “Single-wavelength two-photon excitation-stimulated emission depletion (SW2PE-STED) superresolution imaging,” Proc. Natl. Acad. Sci. U.S.A.109(17), 6390–6393 (2012).
[CrossRef] [PubMed]

C. A. Wurm, D. Neumann, M. A. Lauterbach, B. Harke, A. Egner, S. W. Hell, and S. Jakobs, “Nanoscale distribution of mitochondrial import receptor Tom20 is adjusted to cellular conditions and exhibits an inner-cellular gradient,” Proc. Natl. Acad. Sci. U.S.A.108(33), 13546–13551 (2011).
[CrossRef] [PubMed]

Science

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science320(5873), 246–249 (2008).
[CrossRef] [PubMed]

Other

B. Harke, “3D STED microscopy with pulsed and continuous wave lasers,” Niedersächsische Staats-und Universitätsbibliothek Göttingen (2008).

R. Kasper, B. Harke, C. Forthmann, P. Tinnefeld, S. W. Hell, and M. Sauer, “Single-Molecule STED Microscopy with Photostable Organic Fluorophores,” Small 6, 1379–1384 (2010).

B. Harke, J. Varghese Chacko, C. Canale, H. Haschke, and A. Diaspro, “A novel nanoscopic tool by combining AFM with STED microscopy,” Optical Nanoscopy 1, (2012).

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Figures (5)

Fig. 1
Fig. 1

Simulation on the effect of residuals of fluorescence on STED microscopy performances. Confocal image of a group of subdiffracted beads (upper row). Middle row: STED image of a single point emitter (effective point spread function) by assuming kex = 0.01*ksted and k’sted = 0 (left), k’sted = 0.1*ksted (middle) and k’sted = 0.5*ksted (right). Bottom row: STED image of a group of point emitters in sub-diffraction distance imaged by the above described effective point spread functions. Scale bars: 100 nm.

Fig. 2
Fig. 2

Schematic of the custom made STED microscope. AOM: Acousto optical modulator, PMT: photo multiplier tube, PP: phase plate.

Fig. 3
Fig. 3

(a) Excitation and Emission spectrum of red fluorescent spheres (Invitrogen). (b) fluorescence depletion curve measured in the classical mode (black) and in the modSTED configuration (red) for two different excitation powers of 7.1µW (squares) and 11.4µW (triangles). All power values are measured in front of the back aperture of the objective lens.

Fig. 4
Fig. 4

Comparison of STED images in conventional STED (top row) and ModSTED configuration for different STED powers (4.4 mW; 16.1 mW ; 44.5 mW; 124.5 mW)from left to right) imaging Red Fluorescent 40nm beads with 532nm excitation (11.4 µW) and 642nm STED beam. All power values measured at the back aperture of the objective lens. Confocal standard images (top row, left column) and filtered by lock-in Amplifier (bottom row, left image). Pixel dwell time: 120 µs. Bottom line: line profiles for each STED power value along the path indicated in the image. For better visualization all data were smoothed by a standard Gaussian low pass filter.

Fig. 5
Fig. 5

Imaging of microtubules immunolabeld by the dye ATTO550 and imaged in Confocal mode (a), conventional STED mode (b) and in modSTED (c) configuration. Excitation power 1.8 μW, STED power 180 mW. Pixel dwell time: 100 μs. All data is raw data without any additional post-processing computation.

Equations (3)

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d N 1 ( t ) dt =( k f + k sted ) N 1 ( t )+( k ex +k ' sted ) N 0 ( t )
{ k sted = σ em ( λ sted ) I sted k ' sted = σ ex ( λ sted ) I sted k ex = σ ex ( λ ex ) I ex
N 1 = k ex +k ' sted k f + k sted + k ex +k ' sted

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