Abstract

Stimulated Emission Depletion (STED) microscopy enables subdiffraction resolution in the imaging plane. However, STED's lateral improvement in resolution is generally better than the enhancement in the axial direction. Here, we combine conventional STED superresolution imaging with Double Helix Point Spread Function (PSF) modulation for axial localization with a precision better than the classical Rayleigh limit. To demonstrate the capability of the method we resolve in a STED microscope sub-diffraction fluorescent bead assemblies, and localize them axially with better than 25nm precision. We also show that the same setup allows straightforward implementation of wide field phase contrast by imaging larger beads with spiral and dark field phase filtering.

© 2013 Optical Society of America

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2013 (1)

I. Heller, G. Sitters, O. D. Broekmans, G. Farge, C. Menges, W. Wende, S. W. Hell, E. J. Peterman, and G. J. Wuite, “STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA,” Nat. Methods10(9), 910–916 (2013).
[CrossRef] [PubMed]

2012 (2)

S. Quirin, S. R. P. Pavani, and R. Piestun, “Optimal 3D single-molecule localization for superresolution microscopy with aberrations and engineered point spread functions,” Proc. Natl. Acad. Sci. U. S. A.109(3), 675–679 (2012).
[CrossRef] [PubMed]

T. J. Gould, D. Burke, J. Bewersdorf, and M. J. Booth, “Adaptive optics enables 3D STED microscopy in aberrating specimens,” Opt. Express20(19), 20998–21009 (2012).
[CrossRef] [PubMed]

2011 (2)

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

C. Maurer, A. Jesacher, S. Bernet, and M. Ritsch-Marte, “What spatial light modulators can do for optical microscopy,” Laser Photonics Rev.5(1), 81–101 (2011).
[CrossRef]

2010 (2)

M. Badieirostami, M. D. Lew, M. A. Thompson, and W. E. Moerner, “Three-dimensional localization precision of the double-helix point spread function versus astigmatism and biplane,” Appl. Phys. Lett.97(16), 161103 (2010).
[CrossRef] [PubMed]

G. Grover, S. R. P. Pavani, and R. Piestun, “Performance limits on three-dimensional particle localization in photon-limited microscopy,” Opt. Lett.35(19), 3306–3308 (2010).
[CrossRef] [PubMed]

2009 (4)

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

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U. S. A.106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

K. Y. Han, K. I. Willig, E. Rittweger, F. Jelezko, C. Eggeling, and S. W. Hell, “Three-dimensional stimulated emission depletion microscopy of nitrogen-vacancy centers in diamond using continuous-wave light,” Nano Lett.9(9), 3323–3329 (2009).
[CrossRef] [PubMed]

S. R. P. Pavani, A. Greengard, and R. Piestun, “Three-dimensional localization with nanometer accuracy using a detector-limited double-helix point spread function system,” Appl. Phys. Lett.95(2), 021103 (2009).
[CrossRef]

2008 (6)

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods5(6), 527–529 (2008).
[CrossRef] [PubMed]

S. R. P. Pavani and R. Piestun, “Three dimensional tracking of fluorescent microparticles using a photon-limited double-helix response system,” Opt. Express16(26), 22048–22057 (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]

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

S. R. P. Pavani and R. Piestun, “High-efficiency rotating point spread functions,” Opt. Express16(5), 3484–3489 (2008).
[CrossRef] [PubMed]

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science319(5864), 810–813 (2008).
[CrossRef] [PubMed]

2007 (2)

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

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods4(1), 81–86 (2007).
[CrossRef] [PubMed]

2006 (4)

R. T. Borlinghaus, “MRT letter: high speed scanning has the potential to increase fluorescence yield and to reduce photobleaching,” Microsc. Res. Tech.69(9), 689–692 (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]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J.91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

2003 (1)

1999 (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]

Badieirostami, M.

M. Badieirostami, M. D. Lew, M. A. Thompson, and W. E. Moerner, “Three-dimensional localization precision of the double-helix point spread function versus astigmatism and biplane,” Appl. Phys. Lett.97(16), 161103 (2010).
[CrossRef] [PubMed]

Bates, M.

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science319(5864), 810–813 (2008).
[CrossRef] [PubMed]

Bennett, B. T.

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods5(6), 527–529 (2008).
[CrossRef] [PubMed]

Bernet, S.

C. Maurer, A. Jesacher, S. Bernet, and M. Ritsch-Marte, “What spatial light modulators can do for optical microscopy,” Laser Photonics Rev.5(1), 81–101 (2011).
[CrossRef]

Betzig, E.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Bewersdorf, J.

T. J. Gould, D. Burke, J. Bewersdorf, and M. J. Booth, “Adaptive optics enables 3D STED microscopy in aberrating specimens,” Opt. Express20(19), 20998–21009 (2012).
[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. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods5(6), 527–529 (2008).
[CrossRef] [PubMed]

Biteen, J. S.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U. S. A.106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

Bonifacino, J. S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Booth, M. J.

Borlinghaus, R. T.

R. T. Borlinghaus, “MRT letter: high speed scanning has the potential to increase fluorescence yield and to reduce photobleaching,” Microsc. Res. Tech.69(9), 689–692 (2006).
[CrossRef] [PubMed]

Broekmans, O. D.

I. Heller, G. Sitters, O. D. Broekmans, G. Farge, C. Menges, W. Wende, S. W. Hell, E. J. Peterman, and G. J. Wuite, “STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA,” Nat. Methods10(9), 910–916 (2013).
[CrossRef] [PubMed]

Burke, D.

Davidson, M. W.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Donnert, G.

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

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods4(1), 81–86 (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]

Eggeling, C.

K. Y. Han, K. I. Willig, E. Rittweger, F. Jelezko, C. Eggeling, and S. W. Hell, “Three-dimensional stimulated emission depletion microscopy of nitrogen-vacancy centers in diamond using continuous-wave light,” Nano Lett.9(9), 3323–3329 (2009).
[CrossRef] [PubMed]

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

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods4(1), 81–86 (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]

Egner, A.

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods5(6), 539–544 (2008).
[CrossRef] [PubMed]

Engelhardt, J.

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]

Farge, G.

I. Heller, G. Sitters, O. D. Broekmans, G. Farge, C. Menges, W. Wende, S. W. Hell, E. J. Peterman, and G. J. Wuite, “STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA,” Nat. Methods10(9), 910–916 (2013).
[CrossRef] [PubMed]

Florin, E.-L.

Girirajan, T. P.

S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J.91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

Gould, T. J.

T. J. Gould, D. Burke, J. Bewersdorf, and M. J. Booth, “Adaptive optics enables 3D STED microscopy in aberrating specimens,” Opt. Express20(19), 20998–21009 (2012).
[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. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods5(6), 527–529 (2008).
[CrossRef] [PubMed]

Greengard, A.

S. R. P. Pavani, A. Greengard, and R. Piestun, “Three-dimensional localization with nanometer accuracy using a detector-limited double-helix point spread function system,” Appl. Phys. Lett.95(2), 021103 (2009).
[CrossRef]

Grover, G.

Han, K. Y.

K. Y. Han, K. I. Willig, E. Rittweger, F. Jelezko, C. Eggeling, and S. W. Hell, “Three-dimensional stimulated emission depletion microscopy of nitrogen-vacancy centers in diamond using continuous-wave light,” Nano Lett.9(9), 3323–3329 (2009).
[CrossRef] [PubMed]

Harke, B.

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

Hell, S. W.

I. Heller, G. Sitters, O. D. Broekmans, G. Farge, C. Menges, W. Wende, S. W. Hell, E. J. Peterman, and G. J. Wuite, “STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA,” Nat. Methods10(9), 910–916 (2013).
[CrossRef] [PubMed]

K. Y. Han, K. I. Willig, E. Rittweger, F. Jelezko, C. Eggeling, and S. W. Hell, “Three-dimensional stimulated emission depletion microscopy of nitrogen-vacancy centers in diamond using continuous-wave light,” Nano Lett.9(9), 3323–3329 (2009).
[CrossRef] [PubMed]

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

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]

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

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

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods4(1), 81–86 (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]

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

Heller, I.

I. Heller, G. Sitters, O. D. Broekmans, G. Farge, C. Menges, W. Wende, S. W. Hell, E. J. Peterman, and G. J. Wuite, “STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA,” Nat. Methods10(9), 910–916 (2013).
[CrossRef] [PubMed]

Hess, H. F.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Hess, S. T.

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods5(6), 527–529 (2008).
[CrossRef] [PubMed]

S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J.91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

Huang, B.

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science319(5864), 810–813 (2008).
[CrossRef] [PubMed]

Jahn, R.

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

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

Jakobs, S.

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods5(6), 539–544 (2008).
[CrossRef] [PubMed]

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

Jelezko, F.

K. Y. Han, K. I. Willig, E. Rittweger, F. Jelezko, C. Eggeling, and S. W. Hell, “Three-dimensional stimulated emission depletion microscopy of nitrogen-vacancy centers in diamond using continuous-wave light,” Nano Lett.9(9), 3323–3329 (2009).
[CrossRef] [PubMed]

Jesacher, A.

C. Maurer, A. Jesacher, S. Bernet, and M. Ritsch-Marte, “What spatial light modulators can do for optical microscopy,” Laser Photonics Rev.5(1), 81–101 (2011).
[CrossRef]

Jonás, A.

Juette, M. F.

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods5(6), 527–529 (2008).
[CrossRef] [PubMed]

Kastrup, L.

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

Keller, J.

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

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

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]

Klar, T. A.

Lessard, M. D.

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods5(6), 527–529 (2008).
[CrossRef] [PubMed]

Lew, M. D.

M. Badieirostami, M. D. Lew, M. A. Thompson, and W. E. Moerner, “Three-dimensional localization precision of the double-helix point spread function versus astigmatism and biplane,” Appl. Phys. Lett.97(16), 161103 (2010).
[CrossRef] [PubMed]

Lindwasser, O. W.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Lippincott-Schwartz, J.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Liu, N.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U. S. A.106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

Lord, S. J.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U. S. A.106(9), 2995–2999 (2009).
[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]

Mason, M. D.

S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J.91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

Maurer, C.

C. Maurer, A. Jesacher, S. Bernet, and M. Ritsch-Marte, “What spatial light modulators can do for optical microscopy,” Laser Photonics Rev.5(1), 81–101 (2011).
[CrossRef]

Medda, R.

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

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]

Menges, C.

I. Heller, G. Sitters, O. D. Broekmans, G. Farge, C. Menges, W. Wende, S. W. Hell, E. J. Peterman, and G. J. Wuite, “STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA,” Nat. Methods10(9), 910–916 (2013).
[CrossRef] [PubMed]

Mlodzianoski, M. J.

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods5(6), 527–529 (2008).
[CrossRef] [PubMed]

Moerner, W. E.

M. Badieirostami, M. D. Lew, M. A. Thompson, and W. E. Moerner, “Three-dimensional localization precision of the double-helix point spread function versus astigmatism and biplane,” Appl. Phys. Lett.97(16), 161103 (2010).
[CrossRef] [PubMed]

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U. S. A.106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

Myers, J. R.

Nagpure, B. S.

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods5(6), 527–529 (2008).
[CrossRef] [PubMed]

Olenych, S.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Patterson, G. H.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Pavani, S. R. P.

S. Quirin, S. R. P. Pavani, and R. Piestun, “Optimal 3D single-molecule localization for superresolution microscopy with aberrations and engineered point spread functions,” Proc. Natl. Acad. Sci. U. S. A.109(3), 675–679 (2012).
[CrossRef] [PubMed]

G. Grover, S. R. P. Pavani, and R. Piestun, “Performance limits on three-dimensional particle localization in photon-limited microscopy,” Opt. Lett.35(19), 3306–3308 (2010).
[CrossRef] [PubMed]

S. R. P. Pavani, A. Greengard, and R. Piestun, “Three-dimensional localization with nanometer accuracy using a detector-limited double-helix point spread function system,” Appl. Phys. Lett.95(2), 021103 (2009).
[CrossRef]

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U. S. A.106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

S. R. P. Pavani and R. Piestun, “Three dimensional tracking of fluorescent microparticles using a photon-limited double-helix response system,” Opt. Express16(26), 22048–22057 (2008).
[CrossRef] [PubMed]

S. R. P. Pavani and R. Piestun, “High-efficiency rotating point spread functions,” Opt. Express16(5), 3484–3489 (2008).
[CrossRef] [PubMed]

Peterman, E. J.

I. Heller, G. Sitters, O. D. Broekmans, G. Farge, C. Menges, W. Wende, S. W. Hell, E. J. Peterman, and G. J. Wuite, “STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA,” Nat. Methods10(9), 910–916 (2013).
[CrossRef] [PubMed]

Piestun, R.

S. Quirin, S. R. P. Pavani, and R. Piestun, “Optimal 3D single-molecule localization for superresolution microscopy with aberrations and engineered point spread functions,” Proc. Natl. Acad. Sci. U. S. A.109(3), 675–679 (2012).
[CrossRef] [PubMed]

G. Grover, S. R. P. Pavani, and R. Piestun, “Performance limits on three-dimensional particle localization in photon-limited microscopy,” Opt. Lett.35(19), 3306–3308 (2010).
[CrossRef] [PubMed]

S. R. P. Pavani, A. Greengard, and R. Piestun, “Three-dimensional localization with nanometer accuracy using a detector-limited double-helix point spread function system,” Appl. Phys. Lett.95(2), 021103 (2009).
[CrossRef]

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U. S. A.106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

S. R. P. Pavani and R. Piestun, “Three dimensional tracking of fluorescent microparticles using a photon-limited double-helix response system,” Opt. Express16(26), 22048–22057 (2008).
[CrossRef] [PubMed]

S. R. P. Pavani and R. Piestun, “High-efficiency rotating point spread functions,” Opt. Express16(5), 3484–3489 (2008).
[CrossRef] [PubMed]

Quirin, S.

S. Quirin, S. R. P. Pavani, and R. Piestun, “Optimal 3D single-molecule localization for superresolution microscopy with aberrations and engineered point spread functions,” Proc. Natl. Acad. Sci. U. S. A.109(3), 675–679 (2012).
[CrossRef] [PubMed]

Ritsch-Marte, M.

C. Maurer, A. Jesacher, S. Bernet, and M. Ritsch-Marte, “What spatial light modulators can do for optical microscopy,” Laser Photonics Rev.5(1), 81–101 (2011).
[CrossRef]

Rittweger, E.

K. Y. Han, K. I. Willig, E. Rittweger, F. Jelezko, C. Eggeling, and S. W. Hell, “Three-dimensional stimulated emission depletion microscopy of nitrogen-vacancy centers in diamond using continuous-wave light,” Nano Lett.9(9), 3323–3329 (2009).
[CrossRef] [PubMed]

Rizzoli, S. O.

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

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]

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.

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

Sitters, G.

I. Heller, G. Sitters, O. D. Broekmans, G. Farge, C. Menges, W. Wende, S. W. Hell, E. J. Peterman, and G. J. Wuite, “STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA,” Nat. Methods10(9), 910–916 (2013).
[CrossRef] [PubMed]

Sougrat, R.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Speidel, M.

Thompson, M. A.

M. Badieirostami, M. D. Lew, M. A. Thompson, and W. E. Moerner, “Three-dimensional localization precision of the double-helix point spread function versus astigmatism and biplane,” Appl. Phys. Lett.97(16), 161103 (2010).
[CrossRef] [PubMed]

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U. S. A.106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

Twieg, R. J.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U. S. A.106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

Ullal, C. K.

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

Wang, W.

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science319(5864), 810–813 (2008).
[CrossRef] [PubMed]

Wende, W.

I. Heller, G. Sitters, O. D. Broekmans, G. Farge, C. Menges, W. Wende, S. W. Hell, E. J. Peterman, and G. J. Wuite, “STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA,” Nat. Methods10(9), 910–916 (2013).
[CrossRef] [PubMed]

Westphal, V.

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

Wildanger, D.

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

Willig, K. I.

K. Y. Han, K. I. Willig, E. Rittweger, F. Jelezko, C. Eggeling, and S. W. Hell, “Three-dimensional stimulated emission depletion microscopy of nitrogen-vacancy centers in diamond using continuous-wave light,” Nano Lett.9(9), 3323–3329 (2009).
[CrossRef] [PubMed]

Wuite, G. J.

I. Heller, G. Sitters, O. D. Broekmans, G. Farge, C. Menges, W. Wende, S. W. Hell, E. J. Peterman, and G. J. Wuite, “STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA,” Nat. Methods10(9), 910–916 (2013).
[CrossRef] [PubMed]

Wurm, C. A.

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods5(6), 539–544 (2008).
[CrossRef] [PubMed]

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

Zhuang, X.

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science319(5864), 810–813 (2008).
[CrossRef] [PubMed]

Appl. Phys. Lett. (2)

S. R. P. Pavani, A. Greengard, and R. Piestun, “Three-dimensional localization with nanometer accuracy using a detector-limited double-helix point spread function system,” Appl. Phys. Lett.95(2), 021103 (2009).
[CrossRef]

M. Badieirostami, M. D. Lew, M. A. Thompson, and W. E. Moerner, “Three-dimensional localization precision of the double-helix point spread function versus astigmatism and biplane,” Appl. Phys. Lett.97(16), 161103 (2010).
[CrossRef] [PubMed]

Biophys. J. (2)

S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J.91(11), 4258–4272 (2006).
[CrossRef] [PubMed]

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

J. Microsc. (1)

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

Laser Photonics Rev. (1)

C. Maurer, A. Jesacher, S. Bernet, and M. Ritsch-Marte, “What spatial light modulators can do for optical microscopy,” Laser Photonics Rev.5(1), 81–101 (2011).
[CrossRef]

Microsc. Res. Tech. (1)

R. T. Borlinghaus, “MRT letter: high speed scanning has the potential to increase fluorescence yield and to reduce photobleaching,” Microsc. Res. Tech.69(9), 689–692 (2006).
[CrossRef] [PubMed]

Nano Lett. (2)

K. Y. Han, K. I. Willig, E. Rittweger, F. Jelezko, C. Eggeling, and S. W. Hell, “Three-dimensional stimulated emission depletion microscopy of nitrogen-vacancy centers in diamond using continuous-wave light,” Nano Lett.9(9), 3323–3329 (2009).
[CrossRef] [PubMed]

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

Nat. Methods (4)

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods5(6), 539–544 (2008).
[CrossRef] [PubMed]

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods5(6), 527–529 (2008).
[CrossRef] [PubMed]

G. Donnert, C. Eggeling, and S. W. Hell, “Major signal increase in fluorescence microscopy through dark-state relaxation,” Nat. Methods4(1), 81–86 (2007).
[CrossRef] [PubMed]

I. Heller, G. Sitters, O. D. Broekmans, G. Farge, C. Menges, W. Wende, S. W. Hell, E. J. Peterman, and G. J. Wuite, “STED nanoscopy combined with optical tweezers reveals protein dynamics on densely covered DNA,” Nat. Methods10(9), 910–916 (2013).
[CrossRef] [PubMed]

Opt. Express (4)

Opt. Lett. (3)

Proc. Natl. Acad. Sci. U. S. A. (3)

S. Quirin, S. R. P. Pavani, and R. Piestun, “Optimal 3D single-molecule localization for superresolution microscopy with aberrations and engineered point spread functions,” Proc. Natl. Acad. Sci. U. S. A.109(3), 675–679 (2012).
[CrossRef] [PubMed]

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U. S. A.106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

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

Science (2)

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science319(5864), 810–813 (2008).
[CrossRef] [PubMed]

Other (1)

A. Barsic, G. Grover, and R. Piestun, “Sparse reconstructions of overlapping three-dimensional point spread functions using overcomplete dictionaries,” arXiv:1308.6826 (2013).

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Figures (7)

Fig. 1
Fig. 1

Schematic of the DH assisted STED setup. OPO-optical parametric oscillator; BBO-barium borate doubling crystal; ISO-Faraday isolator; POL-polarizer; SLM-spatial light modulator; BP-band pass fluorescence filter; WP/2-half wave plate; PM-helical phase mask; BS(NP)-non polarizing beam splitter; DM-dichroic mirror; WP/4-quarter wave plate; CP- confocal pinhole; APD-avalanche photodiode; FM-flip mirror. Inserted pictures show the double helix and donut phase fronts assigned respectively by the SLM and the phase plate, scaled from 0 (white) to 2π (black). Two outputs of a femtosecond OPO system are used for STED microscopy (633nm excitation and 770nm depletion). Two imaging paths are available and can be selected with the flip mirror: one classical confocal scanning microscope optical apparatus with an APD and one path with a SLM followed with a CCD allows phase modulation and DH readout.

Fig. 2
Fig. 2

(a) Point spread functions of the microscope recorded with backscattered light from 80nm gold particles. Scale bars are 200nm. Blue and orange color maps are used respectively for excitation and depletion cross sections. (b) Intensity profiles along the dashed color lines on (a). Bottom axis refers to the lateral profile of the excitation and depletion PSFs that are plotted in green and blue respectively. Top axis refers to the axial cross section which is plotted in yellow. Precise alignment and minimal aberration of the PSFs are critical to observe resolution enhancement with STED. The alignment and the quality of the two beams are controlled by scanning gold particles in the focal plane. Confocal FWHM is measured to be 250nm laterally and 730nm axially.

Fig. 3
Fig. 3

Estimation of the lateral resolution of the STED microscope by imaging subdiffraction fluorescent emitters. Transverse images of 20nm beads in glycerol immobilized on a glass slide, with confocal (a) and STED (b) microscope. Scale bars are 500 nm. (c) Bead intensity profile along the yellow dashed line on image (b), fitted with Lorentzian function (red line). The measured FWHM for this bead is 89nm. By averaging over a population of beads, the STED microscope resolution is estimated to be 95nm.

Fig. 4
Fig. 4

Axial localization calibration with DH. (a) Images of a fluorescent bead at different axial positions. (b) Corresponding calibration curve of the inclination of the lobes with respect to the bead axial position. Error bars represent the ± standard deviation of the measured angle over 100 images of fixed beads. (For better visualization of those small intervals error bars are represented by lines filled with 2σ height red transparent rectangles centered on the average position. On the upper right hand corner, the area around the third error bar is magnified). To obtain the calibration curve, a single bead image is recorded at every axial position, and the lobes rotational angle is determined on each image. The accuracy of the measurement is evaluated by repeating several times the same localization event with the depletion beam on and calculating the standard deviation.

Fig. 5
Fig. 5

Three dimensional imaging of a group of 100nm beads immobilized in a PDMS matrix. (a) confocal image (b) corresponding STED image reveals five individual beads (c) Deconvolved STED with numbered cursors on the bead locations and (d) corresponding DH images recorded at these points. (e) 3D representation of the beads position into the dashed boxed delimited on (c) within the focal plane. Scale bars are 500nm. Successive use of STED resolution and DH localization, reveal the three dimensional position of each bead. Although, they appear as a large cluster merged in the focal plane on the confocal image.

Fig. 6
Fig. 6

Photobleaching induced by STED beam. (a) Images of three fluorescent beads represented with the same intensity scaling. From top to bottom: initial confocal scan, consecutive STED image before and after DH localization (around 2s illumination on each bead). (b) Fluorescence photoresistance of 100nm beads under focused light excitation and depletion beam off (yellow), on (blue) and both beams chopped (red). Each curve on (b) is averaged out of 10 single beads. The three scanning images show STED beam induces strong photobleaching during prolonged excitation for the DH axial localization. Fluorescent bead photoresistance can be improved by 20% by chopping the beams.

Fig. 7
Fig. 7

Images of two 1.5µm fluorescent beads immobilized on a glass slide. (a) Wide field fluorescence image. (b) White light illumination image. (c) and (d) Examples of filtering in the Fourier domain: dark field (c) and spiral contrast (d). Insets on the left hand corners represent corresponding phase pattern projected onto the SLM. Scale bars are 1µm.

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