Abstract

We report on fast beam-scanning stimulated-emission-depletion (STED) microscopy in the visible range using for resolution enhancement compact, low cost and turn-key continuous wave (CW) fiber lasers emitting at 592 nm. Spatial resolutions of 35 to 65 nm in the focal plane are shown for various samples including fluorescent nanoparticles, immuno-stained cells with a non-exhaustive selection of 5 commonly used organic fluorescent markers, and living cells expressing the yellow fluorescent protein Citrine. The potential of the straightforward combination of CW-STED and fast beam scanning is illustrated in a movie of the endoplasmic reticulum (ER) of a living cell, composed of 100 frames (6 µm × 12 µm), each of them acquired in a time shorter than 0.2 s.

© 2010 OSA

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  1. 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]
  2. S. W. Hell, “Microscopy and its focal switch,” Nat. Methods 6(1), 24–32 (2009).
    [CrossRef] [PubMed]
  3. T. A. Klar and S. W. Hell, “Subdiffraction resolution in far-field fluorescence microscopy,” Opt. Lett. 24(14), 954–956 (1999).
    [CrossRef]
  4. 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]
  5. 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]
  6. 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]
  7. 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]
  8. 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]
  9. A. C. Meyer, T. Frank, D. Khimich, G. Hoch, D. Riedel, N. M. Chapochnikov, Y. M. Yarin, B. Harke, S. W. Hell, A. Egner, and T. Moser, “Tuning of synapse number, structure and function in the cochlea,” Nat. Neurosci. 12(4), 444–453 (2009).
    [CrossRef] [PubMed]
  10. 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]
  11. 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]
  12. 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]
  13. 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]
  14. 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]
  15. A. Schönle, Imspector Image Acquisition & Analysis Software, v0.1, (2006): http://www.imspector.de
  16. O. Griesbeck, G. S. Baird, R. E. Campbell, D. A. Zacharias, and R. Y. Tsien, “Reducing the environmental sensitivity of yellow fluorescent protein,” J. Biomed. Chem. 276(31), 29188–29194 (2001).
    [CrossRef]
  17. B. Hein, K. I. Willig, and S. W. Hell, “Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell,” Proc. Natl. Acad. Sci. U.S.A. 105(38), 14271–14276 (2008).
    [CrossRef] [PubMed]
  18. 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]
  19. 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]
  20. G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
    [CrossRef]
  21. G. Donnert, C. Eggeling, and S. W. Hell, “Triplet-relaxation microscopy with bunched pulsed excitation,” Photochem. Photobiol. Sci. 8(4), 481–485 (2009).
    [CrossRef] [PubMed]
  22. List of fluorescent dyes used in STED microscopy: http://www.mpibpc.mpg.de/abteilungen/200/STED_Dyes.html

2009 (6)

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]

A. C. Meyer, T. Frank, D. Khimich, G. Hoch, D. Riedel, N. M. Chapochnikov, Y. M. Yarin, B. Harke, S. W. Hell, A. Egner, and T. Moser, “Tuning of synapse number, structure and function in the cochlea,” Nat. Neurosci. 12(4), 444–453 (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]

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

G. Donnert, C. Eggeling, and S. W. Hell, “Triplet-relaxation microscopy with bunched pulsed excitation,” Photochem. Photobiol. Sci. 8(4), 481–485 (2009).
[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]

2008 (5)

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]

B. Hein, K. I. Willig, and S. W. Hell, “Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell,” Proc. Natl. Acad. Sci. U.S.A. 105(38), 14271–14276 (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]

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]

2007 (3)

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]

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]

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (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]

2001 (1)

O. Griesbeck, G. S. Baird, R. E. Campbell, D. A. Zacharias, and R. Y. Tsien, “Reducing the environmental sensitivity of yellow fluorescent protein,” J. Biomed. Chem. 276(31), 29188–29194 (2001).
[CrossRef]

1999 (1)

1994 (1)

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]

Baird, G. S.

O. Griesbeck, G. S. Baird, R. E. Campbell, D. A. Zacharias, and R. Y. Tsien, “Reducing the environmental sensitivity of yellow fluorescent protein,” J. Biomed. Chem. 276(31), 29188–29194 (2001).
[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]

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]

Bückers, J.

Campbell, R. E.

O. Griesbeck, G. S. Baird, R. E. Campbell, D. A. Zacharias, and R. Y. Tsien, “Reducing the environmental sensitivity of yellow fluorescent protein,” J. Biomed. Chem. 276(31), 29188–29194 (2001).
[CrossRef]

Chapochnikov, N. M.

A. C. Meyer, T. Frank, D. Khimich, G. Hoch, D. Riedel, N. M. Chapochnikov, Y. M. Yarin, B. Harke, S. W. Hell, A. Egner, and T. Moser, “Tuning of synapse number, structure and function in the cochlea,” Nat. Neurosci. 12(4), 444–453 (2009).
[CrossRef] [PubMed]

Donnert, G.

G. Donnert, C. Eggeling, and S. W. Hell, “Triplet-relaxation microscopy with bunched pulsed excitation,” Photochem. Photobiol. Sci. 8(4), 481–485 (2009).
[CrossRef] [PubMed]

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
[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]

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, C. Eggeling, and S. W. Hell, “Triplet-relaxation microscopy with bunched pulsed excitation,” Photochem. Photobiol. Sci. 8(4), 481–485 (2009).
[CrossRef] [PubMed]

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
[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.

A. C. Meyer, T. Frank, D. Khimich, G. Hoch, D. Riedel, N. M. Chapochnikov, Y. M. Yarin, B. Harke, S. W. Hell, A. Egner, and T. Moser, “Tuning of synapse number, structure and function in the cochlea,” Nat. Neurosci. 12(4), 444–453 (2009).
[CrossRef] [PubMed]

Frank, T.

A. C. Meyer, T. Frank, D. Khimich, G. Hoch, D. Riedel, N. M. Chapochnikov, Y. M. Yarin, B. Harke, S. W. Hell, A. Egner, and T. Moser, “Tuning of synapse number, structure and function in the cochlea,” Nat. Neurosci. 12(4), 444–453 (2009).
[CrossRef] [PubMed]

Griesbeck, O.

O. Griesbeck, G. S. Baird, R. E. Campbell, D. A. Zacharias, and R. Y. Tsien, “Reducing the environmental sensitivity of yellow fluorescent protein,” J. Biomed. Chem. 276(31), 29188–29194 (2001).
[CrossRef]

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]

Harke, B.

A. C. Meyer, T. Frank, D. Khimich, G. Hoch, D. Riedel, N. M. Chapochnikov, Y. M. Yarin, B. Harke, S. W. Hell, A. Egner, and T. Moser, “Tuning of synapse number, structure and function in the cochlea,” Nat. Neurosci. 12(4), 444–453 (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]

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]

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. Hein, K. I. Willig, and S. W. Hell, “Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell,” Proc. Natl. Acad. Sci. U.S.A. 105(38), 14271–14276 (2008).
[CrossRef] [PubMed]

Hell, S. W.

G. Donnert, C. Eggeling, and S. W. Hell, “Triplet-relaxation microscopy with bunched pulsed excitation,” Photochem. Photobiol. Sci. 8(4), 481–485 (2009).
[CrossRef] [PubMed]

A. C. Meyer, T. Frank, D. Khimich, G. Hoch, D. Riedel, N. M. Chapochnikov, Y. M. Yarin, B. Harke, S. W. Hell, A. Egner, and T. Moser, “Tuning of synapse number, structure and function in the cochlea,” Nat. Neurosci. 12(4), 444–453 (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]

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]

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

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

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]

B. Hein, K. I. Willig, and S. W. Hell, “Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell,” Proc. Natl. Acad. Sci. U.S.A. 105(38), 14271–14276 (2008).
[CrossRef] [PubMed]

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

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

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]

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
[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]

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]

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]

Hoch, G.

A. C. Meyer, T. Frank, D. Khimich, G. Hoch, D. Riedel, N. M. Chapochnikov, Y. M. Yarin, B. Harke, S. W. Hell, A. Egner, and T. Moser, “Tuning of synapse number, structure and function in the cochlea,” Nat. Neurosci. 12(4), 444–453 (2009).
[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.

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]

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]

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]

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.

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]

Khimich, D.

A. C. Meyer, T. Frank, D. Khimich, G. Hoch, D. Riedel, N. M. Chapochnikov, Y. M. Yarin, B. Harke, S. W. Hell, A. Egner, and T. Moser, “Tuning of synapse number, structure and function in the cochlea,” Nat. Neurosci. 12(4), 444–453 (2009).
[CrossRef] [PubMed]

Klar, T. A.

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]

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.

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]

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]

Meyer, A. C.

A. C. Meyer, T. Frank, D. Khimich, G. Hoch, D. Riedel, N. M. Chapochnikov, Y. M. Yarin, B. Harke, S. W. Hell, A. Egner, and T. Moser, “Tuning of synapse number, structure and function in the cochlea,” Nat. Neurosci. 12(4), 444–453 (2009).
[CrossRef] [PubMed]

Moser, T.

A. C. Meyer, T. Frank, D. Khimich, G. Hoch, D. Riedel, N. M. Chapochnikov, Y. M. Yarin, B. Harke, S. W. Hell, A. Egner, and T. Moser, “Tuning of synapse number, structure and function in the cochlea,” Nat. Neurosci. 12(4), 444–453 (2009).
[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]

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]

Riedel, D.

A. C. Meyer, T. Frank, D. Khimich, G. Hoch, D. Riedel, N. M. Chapochnikov, Y. M. Yarin, B. Harke, S. W. Hell, A. Egner, and T. Moser, “Tuning of synapse number, structure and function in the cochlea,” Nat. Neurosci. 12(4), 444–453 (2009).
[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]

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.

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]

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]

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]

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]

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]

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]

Tsien, R. Y.

O. Griesbeck, G. S. Baird, R. E. Campbell, D. A. Zacharias, and R. Y. Tsien, “Reducing the environmental sensitivity of yellow fluorescent protein,” J. Biomed. Chem. 276(31), 29188–29194 (2001).
[CrossRef]

Ullal, C. K.

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.

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]

B. Harke, J. Keller, C. K. Ullal, V. Westphal, A. Schönle, and S. W. Hell, “Resolution scaling in STED microscopy,” Opt. Express 16(6), 4154–4162 (2008).
[CrossRef] [PubMed]

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-rate far-field optical nanoscopy dissects synaptic vesicle movement,” Science 320(5873), 246–249 (2008).
[CrossRef] [PubMed]

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]

Wichmann, J.

Wildanger, D.

Willig, K. I.

B. Hein, K. I. Willig, and S. W. Hell, “Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell,” Proc. Natl. Acad. Sci. U.S.A. 105(38), 14271–14276 (2008).
[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]

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, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (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,” Nature 440(7086), 935–939 (2006).
[CrossRef] [PubMed]

Yarin, Y. M.

A. C. Meyer, T. Frank, D. Khimich, G. Hoch, D. Riedel, N. M. Chapochnikov, Y. M. Yarin, B. Harke, S. W. Hell, A. Egner, and T. Moser, “Tuning of synapse number, structure and function in the cochlea,” Nat. Neurosci. 12(4), 444–453 (2009).
[CrossRef] [PubMed]

Zacharias, D. A.

O. Griesbeck, G. S. Baird, R. E. Campbell, D. A. Zacharias, and R. Y. Tsien, “Reducing the environmental sensitivity of yellow fluorescent protein,” J. Biomed. Chem. 276(31), 29188–29194 (2001).
[CrossRef]

J. Biomed. Chem. (1)

O. Griesbeck, G. S. Baird, R. E. Campbell, D. A. Zacharias, and R. Y. Tsien, “Reducing the environmental sensitivity of yellow fluorescent protein,” J. Biomed. Chem. 276(31), 29188–29194 (2001).
[CrossRef]

Nat. Methods (3)

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

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods 4(1), 81–86 (2007).
[CrossRef]

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

Nat. Neurosci. (1)

A. C. Meyer, T. Frank, D. Khimich, G. Hoch, D. Riedel, N. M. Chapochnikov, Y. M. Yarin, B. Harke, S. W. Hell, A. Egner, and T. Moser, “Tuning of synapse number, structure and function in the cochlea,” Nat. Neurosci. 12(4), 444–453 (2009).
[CrossRef] [PubMed]

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]

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. (2)

Photochem. Photobiol. Sci. (1)

G. Donnert, C. Eggeling, and S. W. Hell, “Triplet-relaxation microscopy with bunched pulsed excitation,” Photochem. Photobiol. Sci. 8(4), 481–485 (2009).
[CrossRef] [PubMed]

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]

B. Hein, K. I. Willig, and S. W. Hell, “Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell,” Proc. Natl. Acad. Sci. U.S.A. 105(38), 14271–14276 (2008).
[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]

Science (1)

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 (2)

A. Schönle, Imspector Image Acquisition & Analysis Software, v0.1, (2006): http://www.imspector.de

List of fluorescent dyes used in STED microscopy: http://www.mpibpc.mpg.de/abteilungen/200/STED_Dyes.html

Supplementary Material (1)

» Media 1: AVI (2574 KB)     

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Figures (4)

Fig. 1
Fig. 1

Experimental setup. The 3 laser beams are injected into single-mode polarization-maintaining fibers (PMF) with achromatic doublets (AD). After collimation, the two STED beams pass through phase masks (PM) and are combined with a polarization beam splitter (PBS). The collinearly aligned beams are then reflected by a dichroic mirror (DM) and circularly polarized by a quarter-waveplate (QWP). They are de-magnified by 2 achromatic doublets, deflected by the scanning mirror (SM) and directed toward the objective lens (OL) by a scan lens (SL) and a tube lens (TL). Before being injected into the microscope, the excitation beam is polarized with a quarter-waveplate and a half-waveplate (HWP) such that its polarization in the back aperture of the objective lens is circular. The fluorescence light passes through notch filters (NF), is injected in a multimode fiber (MF), and finally detected by a photomultiplier (PMT). FP denotes the focal plane.

Fig. 2
Fig. 2

Comparison of confocal and STED images of 20 nm Yellow-green fluorescent particles mounted on a cover glass. (a) and (b) are, respectively, the raw pictures of obtained in confocal and STED modes. (c) and (d) show enlargements of the areas marked with white squares in (a) and (b), respectively. Scale bars correspond to 500 nm. Normalized intensity profiles of the averaged images over more than 10 single bead images taken from the confocal (a) and from the STED (b) pictures, are plotted in (e) in black and red, respectively.

Fig. 3
Fig. 3

Demonstration of similar STED resolution enhancements obtained with the same laser configuration for five commonly used organic fluorescent markers. The five dyes were tested on vimentin filaments in PtK2 cells labeled with primary and secondary antibodies. ai) pictures, with i ranging from 1 to 5, are overview STED images of the vimentin filaments. bi) pictures are enlargements of confocal images, corresponding to the location defined by the white squares in the images shown in the panels ai). The ci) images are the STED counterparts to bi), i.e. the enlargements of the areas marked in panels ai). Finally, di) show normalized intensity line profiles of the filaments marked in ci) where the white bars indicate the location of the black and red profiles extracted from the confocal and STED images, respectively. Scales bars represent 1 µm in images. i = 1 corresponds to Oregon-green 488, i = 2 to Chrome 488, i = 3 to Alexa 488, i = 4 to DY495, and i = 5 to FITC.

Fig. 4
Fig. 4

Intensity-normalized confocal and STED images of the ER of a living PtK2 cell expressing the fluorescent protein Citrine, taken from a time-lapse movie of 100 frames acquired in 190 ms each. The upper left image was recorded in the confocal mode by blocking the STED beam, whereas the other images were recorded in the STED mode. The full movie is available online (Media 1). The fast scanning axis is the vertical axis. Scale bars correspond to 1 µm. Normalized intensity profiles of the images of an ER wall in confocal an STED modes are presented in the inset in black and red, respectively. These profiles correspond to the area located between the arrows in the two upper left frames.

Tables (1)

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Table 1 Fluorescent markers used for CW-STED microscopy at 592 nm

Equations (1)

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d S T E D λ / ( 2 N A 1 + I / I S ) ,

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