Abstract

We show that far-field fluorescence nanoscopy by stimulated emission depletion (STED) can be realized with compact off-the-shelf laser diodes, such as those used in laser pointers and DVDs. A spatial resolution of 40-50 nm is attained by pulsing a 660 nm DVD-diode. The efficacy of these low-cost STED microscopes in biological imaging is demonstrated by differentiating between clusters of the synaptic protein bassoon and transport vesicles in hippocampal neurons, based on the feature diameter. Our results facilitate the implementation of this all-molecular-transition based superresolution method in many applications ranging from nanoscale fluorescence imaging to nanoscale fluorescence sensing.

© 2011 OSA

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    [CrossRef]
  2. 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]
  3. S. W. Hell, “Far-field optical nanoscopy,” Science 316(5828), 1153–1158 (2007).
    [CrossRef] [PubMed]
  4. T. A. Klar and S. W. Hell, “Subdiffraction resolution in far-field fluorescence microscopy,” Opt. Lett. 24(14), 954–956 (1999).
    [CrossRef]
  5. 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]
  6. 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]
  7. E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “STED microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
    [CrossRef]
  8. 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,” Nature 440(7086), 935–939 (2006).
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    [CrossRef]
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef]
  14. M. A. Lauterbach, C. K. Ullal, V. Westphal, and S. W. Hell, “Dynamic imaging of colloidal-crystal nanostructures at 200 frames per second,” Langmuir 26(18), 14400–14404 (2010).
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  17. 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]
  18. 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).
    [PubMed]
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    [CrossRef]
  20. V. N. Rai, M. Shukla, R. K. Khardekar, and H. C. Pant, “A picosecond optical pulse-generator to calibrate the optical streak camera,” Rev. Sci. Instrum. 66(5), 3125–3130 (1995).
    [CrossRef]
  21. 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]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]

2010 (3)

S. Pezzagna, D. Wildanger, P. Mazarov, A. D. Wieck, Y. Sarov, I. Rangelow, B. Naydenov, F. Jelezko, S. W. Hell, and J. Meijer, “Nanoscale engineering and optical addressing of single spins in diamond,” Small 6(19), 2117–2121 (2010).
[CrossRef] [PubMed]

M. A. Lauterbach, C. K. Ullal, V. Westphal, and S. W. Hell, “Dynamic imaging of colloidal-crystal nanostructures at 200 frames per second,” Langmuir 26(18), 14400–14404 (2010).
[CrossRef] [PubMed]

M. Reuss, J. Engelhardt, and S. W. Hell, “Birefringent device converts a standard scanning microscope into a STED microscope that also maps molecular orientation,” Opt. Express 18(2), 1049–1058 (2010).
[CrossRef] [PubMed]

2009 (3)

D. Wildanger, J. Bückers, V. Westphal, S. W. Hell, and L. Kastrup, “A STED microscope aligned by design,” Opt. Express 17(18), 16100–16110 (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]

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “STED microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[CrossRef]

2008 (3)

2007 (3)

V. Westphal, M. A. Lauterbach, A. Di Nicola, and S. W. Hell, “Dynamic far-field fluorescence nanoscopy,” N. J. Phys. 9(12), 435 (2007).
[CrossRef]

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

R. R. Kellner, C. J. Baier, K. I. Willig, S. W. Hell, and F. J. Barrantes, “Nanoscale organization of nicotinic acetylcholine receptors revealed by stimulated emission depletion microscopy,” Neuroscience 144(1), 135–143 (2007).
[CrossRef]

2006 (2)

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,” Nature 440(7086), 935–939 (2006).
[CrossRef] [PubMed]

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

2005 (1)

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 (3)

V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, “Laser-diode-stimulated emission depletion microscopy,” Appl. Phys. Lett. 82(18), 3125–3127 (2003).
[CrossRef]

M. Dyba 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]

M. Shapira, R. G. Zhai, T. Dresbach, T. Bresler, V. I. Torres, E. D. Gundelfinger, N. E. Ziv, and C. C. Garner, “Unitary assembly of presynaptic active zones from Piccolo-Bassoon transport vesicles,” Neuron 38(2), 237–252 (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]

1999 (1)

1995 (1)

V. N. Rai, M. Shukla, R. K. Khardekar, and H. C. Pant, “A picosecond optical pulse-generator to calibrate the optical streak camera,” Rev. Sci. Instrum. 66(5), 3125–3130 (1995).
[CrossRef]

1994 (1)

1873 (1)

E. Abbe, “Beiträge zur theorie des mikroskops und der mikroskopischen wahrnehmung,” Arch. Mikrosc. Anat. Entwicklungsmech. 9, 413–468 (1873).
[CrossRef]

Abbe, E.

E. Abbe, “Beiträge zur theorie des mikroskops und der mikroskopischen wahrnehmung,” Arch. Mikrosc. Anat. Entwicklungsmech. 9, 413–468 (1873).
[CrossRef]

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]

Baier, C. J.

R. R. Kellner, C. J. Baier, K. I. Willig, S. W. Hell, and F. J. Barrantes, “Nanoscale organization of nicotinic acetylcholine receptors revealed by stimulated emission depletion microscopy,” Neuroscience 144(1), 135–143 (2007).
[CrossRef]

Barrantes, F. J.

R. R. Kellner, C. J. Baier, K. I. Willig, S. W. Hell, and F. J. Barrantes, “Nanoscale organization of nicotinic acetylcholine receptors revealed by stimulated emission depletion microscopy,” Neuroscience 144(1), 135–143 (2007).
[CrossRef]

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]

Blanca, C. M.

V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, “Laser-diode-stimulated emission depletion microscopy,” Appl. Phys. Lett. 82(18), 3125–3127 (2003).
[CrossRef]

Bonhoeffer, T.

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

Bresler, T.

M. Shapira, R. G. Zhai, T. Dresbach, T. Bresler, V. I. Torres, E. D. Gundelfinger, N. E. Ziv, and C. C. Garner, “Unitary assembly of presynaptic active zones from Piccolo-Bassoon transport vesicles,” Neuron 38(2), 237–252 (2003).
[CrossRef] [PubMed]

Bückers, J.

Di Nicola, A.

V. Westphal, M. A. Lauterbach, A. Di Nicola, and S. W. Hell, “Dynamic far-field fluorescence nanoscopy,” N. J. Phys. 9(12), 435 (2007).
[CrossRef]

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]

Dresbach, T.

M. Shapira, R. G. Zhai, T. Dresbach, T. Bresler, V. I. Torres, E. D. Gundelfinger, N. E. Ziv, and C. C. Garner, “Unitary assembly of presynaptic active zones from Piccolo-Bassoon transport vesicles,” Neuron 38(2), 237–252 (2003).
[CrossRef] [PubMed]

Dyba, M.

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]

V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, “Laser-diode-stimulated emission depletion microscopy,” Appl. Phys. Lett. 82(18), 3125–3127 (2003).
[CrossRef]

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

Eggeling, 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]

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “STED microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[CrossRef]

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

Egner, A.

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

Engelhardt, J.

Garner, C. C.

M. Shapira, R. G. Zhai, T. Dresbach, T. Bresler, V. I. Torres, E. D. Gundelfinger, N. E. Ziv, and C. C. Garner, “Unitary assembly of presynaptic active zones from Piccolo-Bassoon transport vesicles,” Neuron 38(2), 237–252 (2003).
[CrossRef] [PubMed]

Gundelfinger, E. D.

M. Shapira, R. G. Zhai, T. Dresbach, T. Bresler, V. I. Torres, E. D. Gundelfinger, N. E. Ziv, and C. C. Garner, “Unitary assembly of presynaptic active zones from Piccolo-Bassoon transport vesicles,” Neuron 38(2), 237–252 (2003).
[CrossRef] [PubMed]

Han, K. Y.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “STED microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[CrossRef]

Hein, B.

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]

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

Hell, S. W.

M. Reuss, J. Engelhardt, and S. W. Hell, “Birefringent device converts a standard scanning microscope into a STED microscope that also maps molecular orientation,” Opt. Express 18(2), 1049–1058 (2010).
[CrossRef] [PubMed]

S. Pezzagna, D. Wildanger, P. Mazarov, A. D. Wieck, Y. Sarov, I. Rangelow, B. Naydenov, F. Jelezko, S. W. Hell, and J. Meijer, “Nanoscale engineering and optical addressing of single spins in diamond,” Small 6(19), 2117–2121 (2010).
[CrossRef] [PubMed]

M. A. Lauterbach, C. K. Ullal, V. Westphal, and S. W. Hell, “Dynamic imaging of colloidal-crystal nanostructures at 200 frames per second,” Langmuir 26(18), 14400–14404 (2010).
[CrossRef] [PubMed]

D. Wildanger, J. Bückers, V. Westphal, S. W. Hell, and L. Kastrup, “A STED microscope aligned by design,” Opt. Express 17(18), 16100–16110 (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]

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “STED microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[CrossRef]

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

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).
[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]

V. Westphal, M. A. Lauterbach, A. Di Nicola, and S. W. Hell, “Dynamic far-field fluorescence nanoscopy,” N. J. Phys. 9(12), 435 (2007).
[CrossRef]

R. R. Kellner, C. J. Baier, K. I. Willig, S. W. Hell, and F. J. Barrantes, “Nanoscale organization of nicotinic acetylcholine receptors revealed by stimulated emission depletion microscopy,” Neuroscience 144(1), 135–143 (2007).
[CrossRef]

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

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,” Nature 440(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]

V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, “Laser-diode-stimulated emission depletion microscopy,” Appl. Phys. Lett. 82(18), 3125–3127 (2003).
[CrossRef]

M. Dyba 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]

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

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

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]

Irvine, S. E.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “STED microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[CrossRef]

Jahn, R.

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

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, “STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis,” Nature 440(7086), 935–939 (2006).
[CrossRef] [PubMed]

Jakobs, S.

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]

Jelezko, F.

S. Pezzagna, D. Wildanger, P. Mazarov, A. D. Wieck, Y. Sarov, I. Rangelow, B. Naydenov, F. Jelezko, S. W. Hell, and J. Meijer, “Nanoscale engineering and optical addressing of single spins in diamond,” Small 6(19), 2117–2121 (2010).
[CrossRef] [PubMed]

Kastrup, L.

Keller, J.

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.

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).
[PubMed]

R. R. Kellner, C. J. Baier, K. I. Willig, S. W. Hell, and F. J. Barrantes, “Nanoscale organization of nicotinic acetylcholine receptors revealed by stimulated emission depletion microscopy,” Neuroscience 144(1), 135–143 (2007).
[CrossRef]

Khardekar, R. K.

V. N. Rai, M. Shukla, R. K. Khardekar, and H. C. Pant, “A picosecond optical pulse-generator to calibrate the optical streak camera,” Rev. Sci. Instrum. 66(5), 3125–3130 (1995).
[CrossRef]

Klar, T. A.

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

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

Lauterbach, M. A.

M. A. Lauterbach, C. K. Ullal, V. Westphal, and S. W. Hell, “Dynamic imaging of colloidal-crystal nanostructures at 200 frames per second,” Langmuir 26(18), 14400–14404 (2010).
[CrossRef] [PubMed]

V. Westphal, M. A. Lauterbach, A. Di Nicola, and S. W. Hell, “Dynamic far-field fluorescence nanoscopy,” N. J. Phys. 9(12), 435 (2007).
[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]

Mazarov, P.

S. Pezzagna, D. Wildanger, P. Mazarov, A. D. Wieck, Y. Sarov, I. Rangelow, B. Naydenov, F. Jelezko, S. W. Hell, and J. Meijer, “Nanoscale engineering and optical addressing of single spins in diamond,” Small 6(19), 2117–2121 (2010).
[CrossRef] [PubMed]

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,” Nature 457(7233), 1159–1162 (2009).
[CrossRef]

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

Meijer, J.

S. Pezzagna, D. Wildanger, P. Mazarov, A. D. Wieck, Y. Sarov, I. Rangelow, B. Naydenov, F. Jelezko, S. W. Hell, and J. Meijer, “Nanoscale engineering and optical addressing of single spins in diamond,” Small 6(19), 2117–2121 (2010).
[CrossRef] [PubMed]

Nägerl, U. V.

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

Naydenov, B.

S. Pezzagna, D. Wildanger, P. Mazarov, A. D. Wieck, Y. Sarov, I. Rangelow, B. Naydenov, F. Jelezko, S. W. Hell, and J. Meijer, “Nanoscale engineering and optical addressing of single spins in diamond,” Small 6(19), 2117–2121 (2010).
[CrossRef] [PubMed]

Pant, H. C.

V. N. Rai, M. Shukla, R. K. Khardekar, and H. C. Pant, “A picosecond optical pulse-generator to calibrate the optical streak camera,” Rev. Sci. Instrum. 66(5), 3125–3130 (1995).
[CrossRef]

Pezzagna, S.

S. Pezzagna, D. Wildanger, P. Mazarov, A. D. Wieck, Y. Sarov, I. Rangelow, B. Naydenov, F. Jelezko, S. W. Hell, and J. Meijer, “Nanoscale engineering and optical addressing of single spins in diamond,” Small 6(19), 2117–2121 (2010).
[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,” Nature 457(7233), 1159–1162 (2009).
[CrossRef]

Rai, V. N.

V. N. Rai, M. Shukla, R. K. Khardekar, and H. C. Pant, “A picosecond optical pulse-generator to calibrate the optical streak camera,” Rev. Sci. Instrum. 66(5), 3125–3130 (1995).
[CrossRef]

Rangelow, I.

S. Pezzagna, D. Wildanger, P. Mazarov, A. D. Wieck, Y. Sarov, I. Rangelow, B. Naydenov, F. Jelezko, S. W. Hell, and J. Meijer, “Nanoscale engineering and optical addressing of single spins in diamond,” Small 6(19), 2117–2121 (2010).
[CrossRef] [PubMed]

Rankin, B. R.

Reuss, M.

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]

Rittweger, E.

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “STED microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[CrossRef]

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]

Rizzoli, S. O.

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

K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, and S. W. Hell, “STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis,” Nature 440(7086), 935–939 (2006).
[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]

Sarov, Y.

S. Pezzagna, D. Wildanger, P. Mazarov, A. D. Wieck, Y. Sarov, I. Rangelow, B. Naydenov, F. Jelezko, S. W. Hell, and J. Meijer, “Nanoscale engineering and optical addressing of single spins in diamond,” Small 6(19), 2117–2121 (2010).
[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]

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]

Shapira, M.

M. Shapira, R. G. Zhai, T. Dresbach, T. Bresler, V. I. Torres, E. D. Gundelfinger, N. E. Ziv, and C. C. Garner, “Unitary assembly of presynaptic active zones from Piccolo-Bassoon transport vesicles,” Neuron 38(2), 237–252 (2003).
[CrossRef] [PubMed]

Shukla, M.

V. N. Rai, M. Shukla, R. K. Khardekar, and H. C. Pant, “A picosecond optical pulse-generator to calibrate the optical streak camera,” Rev. Sci. Instrum. 66(5), 3125–3130 (1995).
[CrossRef]

Torres, V. I.

M. Shapira, R. G. Zhai, T. Dresbach, T. Bresler, V. I. Torres, E. D. Gundelfinger, N. E. Ziv, and C. C. Garner, “Unitary assembly of presynaptic active zones from Piccolo-Bassoon transport vesicles,” Neuron 38(2), 237–252 (2003).
[CrossRef] [PubMed]

Ullal, C. K.

M. A. Lauterbach, C. K. Ullal, V. Westphal, and S. W. Hell, “Dynamic imaging of colloidal-crystal nanostructures at 200 frames per second,” Langmuir 26(18), 14400–14404 (2010).
[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]

Westphal, V.

M. A. Lauterbach, C. K. Ullal, V. Westphal, and S. W. Hell, “Dynamic imaging of colloidal-crystal nanostructures at 200 frames per second,” Langmuir 26(18), 14400–14404 (2010).
[CrossRef] [PubMed]

D. Wildanger, J. Bückers, V. Westphal, S. W. Hell, and L. Kastrup, “A STED microscope aligned by design,” Opt. Express 17(18), 16100–16110 (2009).
[CrossRef] [PubMed]

V. Westphal, M. A. Lauterbach, A. Di Nicola, and S. W. Hell, “Dynamic far-field fluorescence nanoscopy,” N. J. Phys. 9(12), 435 (2007).
[CrossRef]

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,” Nature 440(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]

V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, “Laser-diode-stimulated emission depletion microscopy,” Appl. Phys. Lett. 82(18), 3125–3127 (2003).
[CrossRef]

Wichmann, J.

Wieck, A. D.

S. Pezzagna, D. Wildanger, P. Mazarov, A. D. Wieck, Y. Sarov, I. Rangelow, B. Naydenov, F. Jelezko, S. W. Hell, and J. Meijer, “Nanoscale engineering and optical addressing of single spins in diamond,” Small 6(19), 2117–2121 (2010).
[CrossRef] [PubMed]

Wildanger, D.

Willig, K. I.

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

R. R. Kellner, C. J. Baier, K. I. Willig, S. W. Hell, and F. J. Barrantes, “Nanoscale organization of nicotinic acetylcholine receptors revealed by stimulated emission depletion microscopy,” Neuroscience 144(1), 135–143 (2007).
[CrossRef]

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,” Nature 440(7086), 935–939 (2006).
[CrossRef] [PubMed]

Zhai, R. G.

M. Shapira, R. G. Zhai, T. Dresbach, T. Bresler, V. I. Torres, E. D. Gundelfinger, N. E. Ziv, and C. C. Garner, “Unitary assembly of presynaptic active zones from Piccolo-Bassoon transport vesicles,” Neuron 38(2), 237–252 (2003).
[CrossRef] [PubMed]

Ziv, N. E.

M. Shapira, R. G. Zhai, T. Dresbach, T. Bresler, V. I. Torres, E. D. Gundelfinger, N. E. Ziv, and C. C. Garner, “Unitary assembly of presynaptic active zones from Piccolo-Bassoon transport vesicles,” Neuron 38(2), 237–252 (2003).
[CrossRef] [PubMed]

Appl. Opt. (1)

Appl. Phys. Lett. (1)

V. Westphal, C. M. Blanca, M. Dyba, L. Kastrup, and S. W. Hell, “Laser-diode-stimulated emission depletion microscopy,” Appl. Phys. Lett. 82(18), 3125–3127 (2003).
[CrossRef]

Arch. Mikrosc. Anat. Entwicklungsmech. (1)

E. Abbe, “Beiträge zur theorie des mikroskops und der mikroskopischen wahrnehmung,” Arch. Mikrosc. Anat. Entwicklungsmech. 9, 413–468 (1873).
[CrossRef]

Langmuir (1)

M. A. Lauterbach, C. K. Ullal, V. Westphal, and S. W. Hell, “Dynamic imaging of colloidal-crystal nanostructures at 200 frames per second,” Langmuir 26(18), 14400–14404 (2010).
[CrossRef] [PubMed]

N. J. Phys. (1)

V. Westphal, M. A. Lauterbach, A. Di Nicola, and S. W. Hell, “Dynamic far-field fluorescence nanoscopy,” N. J. Phys. 9(12), 435 (2007).
[CrossRef]

Nat. Photonics (1)

E. Rittweger, K. Y. Han, S. E. Irvine, C. Eggeling, and S. W. Hell, “STED microscopy reveals crystal colour centres with nanometric resolution,” Nat. Photonics 3(3), 144–147 (2009).
[CrossRef]

Nature (2)

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,” Nature 440(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,” Nature 457(7233), 1159–1162 (2009).
[CrossRef]

Neuron (1)

M. Shapira, R. G. Zhai, T. Dresbach, T. Bresler, V. I. Torres, E. D. Gundelfinger, N. E. Ziv, and C. C. Garner, “Unitary assembly of presynaptic active zones from Piccolo-Bassoon transport vesicles,” Neuron 38(2), 237–252 (2003).
[CrossRef] [PubMed]

Neuroscience (1)

R. R. Kellner, C. J. Baier, K. I. Willig, S. W. Hell, and F. J. Barrantes, “Nanoscale organization of nicotinic acetylcholine receptors revealed by stimulated emission depletion microscopy,” Neuroscience 144(1), 135–143 (2007).
[CrossRef]

Opt. Express (3)

Opt. Lett. (3)

Phys. Rev. Lett. (1)

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. (3)

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]

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

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]

Rev. Sci. Instrum. (1)

V. N. Rai, M. Shukla, R. K. Khardekar, and H. C. Pant, “A picosecond optical pulse-generator to calibrate the optical streak camera,” Rev. Sci. Instrum. 66(5), 3125–3130 (1995).
[CrossRef]

Science (1)

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

Small (1)

S. Pezzagna, D. Wildanger, P. Mazarov, A. D. Wieck, Y. Sarov, I. Rangelow, B. Naydenov, F. Jelezko, S. W. Hell, and J. Meijer, “Nanoscale engineering and optical addressing of single spins in diamond,” Small 6(19), 2117–2121 (2010).
[CrossRef] [PubMed]

Other (1)

A. Fejtova and E. D. Gundelfinger, “Molecular organization and assembly of the presynaptic active zone of neurotransmitter release,” in Results and Problems in Cell Differentiation (Springer-Verlag, 2006), pp. 49–68.
[PubMed]

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Figures (4)

Fig. 1
Fig. 1

Employing a DVD laser diode as light source for STED. Setup including the schematic diagram of the electrical driver circuit for the laser diode and the intensity profile of the generated pulses. T: transistor, C: capacitor, PG: pulse generator, GND: ground; DM: dichroic mirror; APD: avalanche photodiode.

Fig. 2
Fig. 2

To demonstrate STED an image of fluorescent beads was recorded at constant excitation but modulated STED-beam (left). The corresponding fluorescence intensity profile of the image (right) shows that when applying STED the fluorescence signal drops down to < 10%.

Fig. 3
Fig. 3

STED imaging with the compact and simple laser diode: (a) confocal image of Nile red fluorescence beads and corresponding STED images ((b) raw data and (c) linear deconvolution) (a-1), (b-1) Magnified view of the boxed region in (a) and (b). (d) The corresponding line profiles of confocal (green) and STED mode (red) highlight a more than 4-fold resolution gain. Bar size corresponds to 250 nm.

Fig. 4
Fig. 4

Presynaptic active zone protein Bassoon in hippocampal-neurons. (a),(d) Confocal image, (b), (e) STED raw data, and (c), (f) STED data linearly deconvolved. Magnified views of the boxed region in (d) and (f). The line profiles through these corresponding structures illustrate the resolution gain in the STED mode. Through the enhanced resolution of STED the active zone (AZ) and bassoon/piccolo transport vesicles (PTVs) can be distinguished by their different diameters. Bar size corresponds to 500 nm.

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