Abstract

We introduce a parallelized STED microscope featuring m = 4 pairs of scanning excitation and STED beams, providing m-fold increased imaging speed of a given sample area, while maintaining basically all of the advantages of single beam scanning. Requiring only a single laser source and fiber input, the setup is inherently aligned both spatially and temporally. Given enough laser power, the design is readily scalable to higher degrees of parallelization m.

© 2011 OSA

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    [CrossRef] [PubMed]
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    [CrossRef]
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  7. M. J. Rust, M. Bates, and X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
  19. J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express 15(6), 3361–3371 (2007).
    [CrossRef] [PubMed]
  20. M. A. Schwentker, H. Bock, M. Hofmann, S. Jakobs, J. Bewersdorf, C. Eggeling, and S. W. Hell, “Wide-field subdiffraction RESOLFT microscopy using fluorescent protein photoswitching,” Microsc. Res. Tech. 70(3), 269–280 (2007).
    [CrossRef] [PubMed]
  21. 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]
  22. G. Wong, R. Pilkington, and A. R. Harvey, “Achromatization of Wollaston polarizing beam splitters,” Opt. Lett. 36(8), 1332–1334 (2011).
    [CrossRef] [PubMed]
  23. 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]
  24. 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]
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    [CrossRef] [PubMed]

2011 (1)

2010 (2)

2009 (2)

2007 (4)

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

M. A. Schwentker, H. Bock, M. Hofmann, S. Jakobs, J. Bewersdorf, C. Eggeling, and S. W. Hell, “Wide-field subdiffraction RESOLFT microscopy using fluorescent protein photoswitching,” Microsc. Res. Tech. 70(3), 269–280 (2007).
[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] [PubMed]

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

2006 (4)

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,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

M. J. Rust, M. Bates, and X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

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

2005 (1)

M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A. 102(37), 13081–13086 (2005).
[CrossRef] [PubMed]

2003 (2)

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

S. W. Hell, S. Jakobs, and L. Kastrup, “Imaging and writing at the nanoscale with focused visible light through saturable optical transitions,” Appl. Phys., A Mater. Sci. Process. 77(7), 859–860 (2003).
[CrossRef]

2002 (1)

2001 (1)

T. A. Klar, E. Engel, and S. W. Hell, “Breaking Abbe’s diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes,” Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 64(6), 066613 (2001).
[CrossRef] [PubMed]

2000 (2)

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]

M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000).
[CrossRef] [PubMed]

1999 (1)

1996 (1)

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, “High-speed confocal fluorescence microscopy using a Nipkow scanner with microlenses for 3D-imaging of single fluorescent molecule in real time,” Bioimaging 4, 52–62 (1996).

1995 (1)

S. W. Hell and M. Kroug, “Ground-state depletion fluorescence microscopy, a concept for breaking the diffraction resolution limit,” Appl. Phys. B 60(5), 495–497 (1995).
[CrossRef]

1994 (1)

Abe, M.

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, “High-speed confocal fluorescence microscopy using a Nipkow scanner with microlenses for 3D-imaging of single fluorescent molecule in real time,” Bioimaging 4, 52–62 (1996).

Babcock, H.

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

Bates, M.

M. J. Rust, M. Bates, and X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

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,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Bewersdorf, J.

M. A. Schwentker, H. Bock, M. Hofmann, S. Jakobs, J. Bewersdorf, C. Eggeling, and S. W. Hell, “Wide-field subdiffraction RESOLFT microscopy using fluorescent protein photoswitching,” Microsc. Res. Tech. 70(3), 269–280 (2007).
[CrossRef] [PubMed]

Bock, H.

M. A. Schwentker, H. Bock, M. Hofmann, S. Jakobs, J. Bewersdorf, C. Eggeling, and S. W. Hell, “Wide-field subdiffraction RESOLFT microscopy using fluorescent protein photoswitching,” Microsc. Res. Tech. 70(3), 269–280 (2007).
[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,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Bückers, J.

Cremer, C.

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,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Donnert, G.

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] [PubMed]

Dyba, M.

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.

M. A. Schwentker, H. Bock, M. Hofmann, S. Jakobs, J. Bewersdorf, C. Eggeling, and S. W. Hell, “Wide-field subdiffraction RESOLFT microscopy using fluorescent protein photoswitching,” Microsc. Res. Tech. 70(3), 269–280 (2007).
[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] [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]

Engel, E.

T. A. Klar, E. Engel, and S. W. Hell, “Breaking Abbe’s diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes,” Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 64(6), 066613 (2001).
[CrossRef] [PubMed]

Engelhardt, J.

Girirajan, T. P. K.

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

Gustafsson, M. G. L.

M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A. 102(37), 13081–13086 (2005).
[CrossRef] [PubMed]

M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000).
[CrossRef] [PubMed]

Harvey, A. R.

Heintzmann, R.

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]

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]

M. A. Schwentker, H. Bock, M. Hofmann, S. Jakobs, J. Bewersdorf, C. Eggeling, and S. W. Hell, “Wide-field subdiffraction RESOLFT microscopy using fluorescent protein photoswitching,” Microsc. Res. Tech. 70(3), 269–280 (2007).
[CrossRef] [PubMed]

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

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

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] [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]

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

S. W. Hell, S. Jakobs, and L. Kastrup, “Imaging and writing at the nanoscale with focused visible light through saturable optical transitions,” Appl. Phys., A Mater. Sci. Process. 77(7), 859–860 (2003).
[CrossRef]

T. A. Klar, E. Engel, and S. W. Hell, “Breaking Abbe’s diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes,” Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 64(6), 066613 (2001).
[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] [PubMed]

S. W. Hell and M. Kroug, “Ground-state depletion fluorescence microscopy, a concept for breaking the diffraction resolution limit,” Appl. Phys. B 60(5), 495–497 (1995).
[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]

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,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Hess, S. T.

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

Hofmann, M.

M. A. Schwentker, H. Bock, M. Hofmann, S. Jakobs, J. Bewersdorf, C. Eggeling, and S. W. Hell, “Wide-field subdiffraction RESOLFT microscopy using fluorescent protein photoswitching,” Microsc. Res. Tech. 70(3), 269–280 (2007).
[CrossRef] [PubMed]

Huang, B.

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

Ichihara, A.

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, “High-speed confocal fluorescence microscopy using a Nipkow scanner with microlenses for 3D-imaging of single fluorescent molecule in real time,” Bioimaging 4, 52–62 (1996).

Isozaki, K.

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, “High-speed confocal fluorescence microscopy using a Nipkow scanner with microlenses for 3D-imaging of single fluorescent molecule in real time,” Bioimaging 4, 52–62 (1996).

Jahn, R.

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.

M. A. Schwentker, H. Bock, M. Hofmann, S. Jakobs, J. Bewersdorf, C. Eggeling, and S. W. Hell, “Wide-field subdiffraction RESOLFT microscopy using fluorescent protein photoswitching,” Microsc. Res. Tech. 70(3), 269–280 (2007).
[CrossRef] [PubMed]

S. W. Hell, S. Jakobs, and L. Kastrup, “Imaging and writing at the nanoscale with focused visible light through saturable optical transitions,” Appl. Phys., A Mater. Sci. Process. 77(7), 859–860 (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]

Jovin, T. M.

Kastrup, L.

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, S. Jakobs, and L. Kastrup, “Imaging and writing at the nanoscale with focused visible light through saturable optical transitions,” Appl. Phys., A Mater. Sci. Process. 77(7), 859–860 (2003).
[CrossRef]

Keller, J.

Klar, T. A.

T. A. Klar, E. Engel, and S. W. Hell, “Breaking Abbe’s diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes,” Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 64(6), 066613 (2001).
[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] [PubMed]

Kosugi, Y.

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, “High-speed confocal fluorescence microscopy using a Nipkow scanner with microlenses for 3D-imaging of single fluorescent molecule in real time,” Bioimaging 4, 52–62 (1996).

Kroug, M.

S. W. Hell and M. Kroug, “Ground-state depletion fluorescence microscopy, a concept for breaking the diffraction resolution limit,” Appl. Phys. B 60(5), 495–497 (1995).
[CrossRef]

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,” Science 313(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,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Mason, M. D.

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

Mikuriya, K.

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, “High-speed confocal fluorescence microscopy using a Nipkow scanner with microlenses for 3D-imaging of single fluorescent molecule in real time,” Bioimaging 4, 52–62 (1996).

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,” Science 313(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,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Pilkington, R.

Reuss, M.

Rizzoli, S. O.

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]

Rust, M. J.

M. J. Rust, M. Bates, and X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

Schönle, A.

Schwentker, M. A.

M. A. Schwentker, H. Bock, M. Hofmann, S. Jakobs, J. Bewersdorf, C. Eggeling, and S. W. Hell, “Wide-field subdiffraction RESOLFT microscopy using fluorescent protein photoswitching,” Microsc. Res. Tech. 70(3), 269–280 (2007).
[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,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Sugiyama, Y.

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, “High-speed confocal fluorescence microscopy using a Nipkow scanner with microlenses for 3D-imaging of single fluorescent molecule in real time,” Bioimaging 4, 52–62 (1996).

Tanaami, T.

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, “High-speed confocal fluorescence microscopy using a Nipkow scanner with microlenses for 3D-imaging of single fluorescent molecule in real time,” Bioimaging 4, 52–62 (1996).

Uemura, I.

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, “High-speed confocal fluorescence microscopy using a Nipkow scanner with microlenses for 3D-imaging of single fluorescent molecule in real time,” Bioimaging 4, 52–62 (1996).

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]

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]

Wichmann, J.

Wildanger, D.

Willig, K. I.

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

Wong, G.

Zhuang, X.

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

Zhuang, X. W.

M. J. Rust, M. Bates, and X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

Appl. Phys. B (1)

S. W. Hell and M. Kroug, “Ground-state depletion fluorescence microscopy, a concept for breaking the diffraction resolution limit,” Appl. Phys. B 60(5), 495–497 (1995).
[CrossRef]

Appl. Phys., A Mater. Sci. Process. (1)

S. W. Hell, S. Jakobs, and L. Kastrup, “Imaging and writing at the nanoscale with focused visible light through saturable optical transitions,” Appl. Phys., A Mater. Sci. Process. 77(7), 859–860 (2003).
[CrossRef]

Bioimaging (1)

A. Ichihara, T. Tanaami, K. Isozaki, Y. Sugiyama, Y. Kosugi, K. Mikuriya, M. Abe, and I. Uemura, “High-speed confocal fluorescence microscopy using a Nipkow scanner with microlenses for 3D-imaging of single fluorescent molecule in real time,” Bioimaging 4, 52–62 (1996).

Biophys. J. (1)

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

Cell (1)

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

J. Microsc. (1)

M. G. L. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A (1)

Microsc. Res. Tech. (1)

M. A. Schwentker, H. Bock, M. Hofmann, S. Jakobs, J. Bewersdorf, C. Eggeling, and S. W. Hell, “Wide-field subdiffraction RESOLFT microscopy using fluorescent protein photoswitching,” Microsc. Res. Tech. 70(3), 269–280 (2007).
[CrossRef] [PubMed]

Nat. Biotechnol. (1)

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

Nat. Methods (3)

M. J. Rust, M. Bates, and X. W. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[CrossRef] [PubMed]

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] [PubMed]

Nature (1)

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]

Opt. Express (3)

Opt. Lett. (3)

Phys. Rev. E Stat. Nonlin. Soft Matter Phys. (1)

T. A. Klar, E. Engel, and S. W. Hell, “Breaking Abbe’s diffraction resolution limit in fluorescence microscopy with stimulated emission depletion beams of various shapes,” Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 64(6), 066613 (2001).
[CrossRef] [PubMed]

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

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]

M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A. 102(37), 13081–13086 (2005).
[CrossRef] [PubMed]

Science (2)

S. W. Hell, “Far-field optical nanoscopy,” Science 316(5828), 1153–1158 (2007).
[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,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Other (1)

S. W. Hell, “Increasing the resolution of far-field fluorescence light microscopy by point-spread-function engineering,” in Topics in Fluorescence Spectroscopy, J. R. Lakowicz, ed. (Plenum Press, New York, 1997), pp. 361–422.

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Figures (6)

Fig. 1
Fig. 1

Parallelized STED microscope: A stack of Wollaston prisms (W1,W2) are used for generating multiple foci and a segmented waveplate (SWP) for shaping the beams to either excitation spots or doughnut-shaped STED beams (Inset: Image Plane). Unpolarized excitation (635 nm) and STED (745 nm) originating from outputs pumped by a common laser are combined using a dichroic mirror (DC) and coupled into a common single-mode-fiber after adjusting timing by inserting an optical delay (not shown). Both unpolarized output beams are split into four polarized beamlets in the conjugated backfocal plane (Inset: Conjugated Backfocal Plane; arrows indicate linear polarization orientation of the individual beamlets). Depending on the available laser power, this configuration is scalable to any power of 2 by adding further Wollaston prisms. All beamlets are then reflected by a bandpass filter (BP) and focused into a scanning device (Quadscanner), consisting of four motorized mirrors, placed in the conjugated image plane (Inset: Conjugated Image Plane). The first pair of of mirrors induces a lateral displacement in one direction (X-Scan) while the second pair displaces the beams in the perpendicular direction (Y-Scan). All beamlets are circularized using a quarter-wave plate (QWP) to avoid a bias on STED induced by molecular orientation before passing through the SWP which acts as beamshaping device (Inset: Separating element). The fluorescence passes back through the scanner and bandpass filter before being focused onto a mirrored pyramid which splits the fluorescence originating from the four focal volumes. Each fluorescent signal is focused onto a single-photon counting module (APD).

Fig. 2
Fig. 2

Beamshaping by the segmented waveplate in combination with two Wollaston prisms. Left: STED (top) and excitation (bottom) spots are arranged in the rhombus-like pattern when focusing onto a camera in the backfocal plane (scale bar: 1 μm). Top right: Focal intensity distributions of one of the four parallelized excitation (green) and STED (red) beam pairs. The segmented wave plate converts the 745 nm STED beam into a doughnut and the 633 nm beam into a regular spot (Scale bar: 500nm). Bottom right: line profiles of excitation (green) and STED (red) focal spots along the direction indicated by the arrows on the top image.

Fig. 3
Fig. 3

Fluorescent beads measured using parallelized confocal (top) and STED (center) by either adding overlapping frames (left) or stitching non-overlapping frames together (right). Scale bars: 500 nm. Both scanning schemes offer a fourfold faster acquisition of superresolving STED microscopy. Bottom: Line profiles across the beads indicated by the white arrows above. All images represent raw data and were obtained using pixel dwell times of 50/200 μsec (overlay/stitched) and a pixel size of 20 nm.

Fig. 4
Fig. 4

Left: Confocal overview and magnified region (inset) of KK114-labelled tubulin strands in a fixed mammalian (PtK2) cell. Right: Magnified STED images of the four parallelized detection channels 1-4, each corresponding to the confocal region. Note that all STED channels show identical features and that four parallel tubulin fibers (arrows) can be observed using STED which cannot be resolved using confocal microscopy. The confocal images have been recorderd using one of the detection channels. The other three channels provide similar images (not shown). All images represent raw data and were obtained using a pixel dwell time of 100 μsec and pixel size of 20 nm. Scale bars: 1 μm / 10 μm (Magnifications/Overview).

Fig. 5
Fig. 5

Top left: Confocal overview of KK114-labelled vimentin fibers in mammalian (PtK2) cells. Scale bar: 5 μm Right: Confocal (top) and STED (bottom) images of area indicated on the left (white square) using a stitched scanning scheme. Note the Rhombus-like stitching pattern. To avoid overlapping of frames, overlapped regions were considered only once. Scale bars: 1 μm. Bottom left: Large single channel STED image following STED and confocal stitched images for comparison. The other detection channels show similar images (not shown). No significant disparities between stitched and single frames images can be observed when following individual vimentin fibers along multiple frames. All images represent raw data and were obtained using a pixel dwell time of 20 μsec and pixel size of 20 nm. Scale bar: 1 μm.

Fig. 6
Fig. 6

Comparison of single STED image (left) at 200 μsec pixel dwell time of the first spot passing the sample (to avoid comparing a bleached image) and by adding the four detection channels at a pixel dwell time of 50 μsec (right) of Atto647N-labelled tubulin in PtK2 cells. Scale bars: 1 μm. All images represent raw data and were obtained using a pixel size of 20 nm.

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