Abstract

Despite the need for isotropic optical resolution in a growing number of applications, the majority of super-resolution fluorescence microscopy setups still do not attain an axial resolution comparable to that in the lateral dimensions. Three-dimensional (3D) nanoscopy implementations that employ only a single objective lens typically feature a trade-off between axial and lateral resolution. 4Pi arrangements, in which the sample is illuminated coherently through two opposing lenses, have proven their potential for rendering the resolution isotropic. However, instrument complexity due to a large number of alignment parameters has so far thwarted the dissemination of this approach. Here, we present a 4Pi-STED setup combination, also called isoSTED nanoscope, where the STED and excitation beams are intrinsically co-aligned. A highly robust and convenient 4Pi cavity allows easy handling without the need for readjustments during imaging experiments.

© 2015 Optical Society of America

Full Article  |  PDF Article
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References

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2015 (1)

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349(6251), aab3500 (2015).
[Crossref] [PubMed]

2014 (1)

F. Görlitz, P. Hoyer, H. Falk, L. Kastrup, J. Engelhardt, and S. W. Hell, “A STED microscope designed for routine biomedical applications,” Prog. Electromagnetics Res. 147, 57–68 (2014).
[Crossref]

2012 (1)

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A. 109(3), E135–E143 (2012).
[Crossref] [PubMed]

2011 (1)

D. Aquino, A. Schönle, C. Geisler, C. V. Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, and A. Egner, “Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores,” Nat. Methods 8(4), 353–359 (2011).
[Crossref] [PubMed]

2010 (1)

2009 (5)

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]

R. Schmidt, C. A. Wurm, A. Punge, A. Egner, S. Jakobs, and S. W. Hell, “Mitochondrial cristae revealed with focused light,” Nano Lett. 9(6), 2508–2510 (2009).
[Crossref] [PubMed]

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (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]

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

2008 (4)

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319(5864), 810–813 (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]

M. C. Lang, T. Staudt, J. Engelhardt, and S. W. Hell, “4Pi microscopy with negligible sidelobes,” New J. Phys. 10(4), 043041 (2008).
[Crossref]

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. Methods 5(6), 539–544 (2008).
[Crossref] [PubMed]

2007 (5)

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]

2004 (1)

H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, “Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy,” Biophys. J. 87(6), 4146–4152 (2004).
[Crossref] [PubMed]

2002 (1)

M. Dyba and S. W. Hell, “Focal spots of size lambda/23 open up far-field fluorescence microscopy at 33 nm axial resolution,” Phys. Rev. Lett. 88(16), 163901 (2002).
[Crossref] [PubMed]

2000 (1)

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

1999 (1)

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, “I5M: 3D widefield light microscopy with better than 100 nm axial resolution,” J. Microsc. 195(1), 10–16 (1999).
[Crossref] [PubMed]

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)

1992 (1)

1959 (2)

E. Wolf, “Electromagnetic diffraction in optical systems I. An integral representation of the image field,” Proc. R. Soc. Lond. A Math. Phys. Sci. 253(1274), 349–357 (1959).
[Crossref]

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A Math. Phys. Sci. 253(1274), 358–379 (1959).
[Crossref]

Agard, D. A.

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, “I5M: 3D widefield light microscopy with better than 100 nm axial resolution,” J. Microsc. 195(1), 10–16 (1999).
[Crossref] [PubMed]

Aquino, D.

D. Aquino, A. Schönle, C. Geisler, C. V. Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, and A. Egner, “Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores,” Nat. Methods 8(4), 353–359 (2011).
[Crossref] [PubMed]

Baird, M. A.

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349(6251), aab3500 (2015).
[Crossref] [PubMed]

Bates, M.

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319(5864), 810–813 (2008).
[Crossref] [PubMed]

Beach, J. R.

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349(6251), aab3500 (2015).
[Crossref] [PubMed]

Betzig, E.

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349(6251), aab3500 (2015).
[Crossref] [PubMed]

Bewersdorf, J.

H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, “Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy,” Biophys. J. 87(6), 4146–4152 (2004).
[Crossref] [PubMed]

Bückers, J.

Chen, B.-C.

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349(6251), aab3500 (2015).
[Crossref] [PubMed]

Davidson, M. W.

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349(6251), aab3500 (2015).
[Crossref] [PubMed]

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A. 109(3), E135–E143 (2012).
[Crossref] [PubMed]

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[Crossref] [PubMed]

Dedecker, P.

Dyba, M.

M. Dyba and S. W. Hell, “Focal spots of size lambda/23 open up far-field fluorescence microscopy at 33 nm axial resolution,” Phys. Rev. Lett. 88(16), 163901 (2002).
[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]

Egner, A.

D. Aquino, A. Schönle, C. Geisler, C. V. Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, and A. Egner, “Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores,” Nat. Methods 8(4), 353–359 (2011).
[Crossref] [PubMed]

R. Schmidt, C. A. Wurm, A. Punge, A. Egner, S. Jakobs, and S. W. Hell, “Mitochondrial cristae revealed with focused light,” Nano Lett. 9(6), 2508–2510 (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. Methods 5(6), 539–544 (2008).
[Crossref] [PubMed]

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

Enderlein, J.

Engelhardt, J.

F. Görlitz, P. Hoyer, H. Falk, L. Kastrup, J. Engelhardt, and S. W. Hell, “A STED microscope designed for routine biomedical applications,” Prog. Electromagnetics Res. 147, 57–68 (2014).
[Crossref]

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]

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. Methods 5(6), 539–544 (2008).
[Crossref] [PubMed]

M. C. Lang, T. Staudt, J. Engelhardt, and S. W. Hell, “4Pi microscopy with negligible sidelobes,” New J. Phys. 10(4), 043041 (2008).
[Crossref]

T. Staudt, M. C. Lang, R. Medda, J. Engelhardt, and S. W. Hell, “2,2′-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy,” Microsc. Res. Tech. 70(1), 1–9 (2007).
[Crossref] [PubMed]

M. C. Lang, J. Engelhardt, and S. W. Hell, “4Pi microscopy with linear fluorescence excitation,” Opt. Lett. 32(3), 259–261 (2007).
[Crossref] [PubMed]

M. Lang, T. Müller, J. Engelhardt, and S. W. Hell, “4Pi microscopy of type A with 1-photon excitation in biological fluorescence imaging,” Opt. Express 15(5), 2459–2467 (2007).
[Crossref] [PubMed]

H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, “Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy,” Biophys. J. 87(6), 4146–4152 (2004).
[Crossref] [PubMed]

Falk, H.

F. Görlitz, P. Hoyer, H. Falk, L. Kastrup, J. Engelhardt, and S. W. Hell, “A STED microscope designed for routine biomedical applications,” Prog. Electromagnetics Res. 147, 57–68 (2014).
[Crossref]

Fetter, R. D.

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[Crossref] [PubMed]

Galbraith, C. G.

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[Crossref] [PubMed]

Galbraith, J. A.

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[Crossref] [PubMed]

Geisler, C.

D. Aquino, A. Schönle, C. Geisler, C. V. Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, and A. Egner, “Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores,” Nat. Methods 8(4), 353–359 (2011).
[Crossref] [PubMed]

Gillette, J. M.

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[Crossref] [PubMed]

Görlitz, F.

F. Görlitz, P. Hoyer, H. Falk, L. Kastrup, J. Engelhardt, and S. W. Hell, “A STED microscope designed for routine biomedical applications,” Prog. Electromagnetics Res. 147, 57–68 (2014).
[Crossref]

Gugel, H.

H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, “Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy,” Biophys. J. 87(6), 4146–4152 (2004).
[Crossref] [PubMed]

Gustafsson, M. G.

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A. 109(3), E135–E143 (2012).
[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, D. A. Agard, and J. W. Sedat, “I5M: 3D widefield light microscopy with better than 100 nm axial resolution,” J. Microsc. 195(1), 10–16 (1999).
[Crossref] [PubMed]

Hammer, J. A.

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349(6251), aab3500 (2015).
[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.

Hell, S. W.

F. Görlitz, P. Hoyer, H. Falk, L. Kastrup, J. Engelhardt, and S. W. Hell, “A STED microscope designed for routine biomedical applications,” Prog. Electromagnetics Res. 147, 57–68 (2014).
[Crossref]

D. Aquino, A. Schönle, C. Geisler, C. V. Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, and A. Egner, “Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores,” Nat. Methods 8(4), 353–359 (2011).
[Crossref] [PubMed]

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

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

R. Schmidt, C. A. Wurm, A. Punge, A. Egner, S. Jakobs, and S. W. Hell, “Mitochondrial cristae revealed with focused light,” Nano Lett. 9(6), 2508–2510 (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]

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]

M. C. Lang, T. Staudt, J. Engelhardt, and S. W. Hell, “4Pi microscopy with negligible sidelobes,” New J. Phys. 10(4), 043041 (2008).
[Crossref]

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. Methods 5(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]

M. Lang, T. Müller, J. Engelhardt, and S. W. Hell, “4Pi microscopy of type A with 1-photon excitation in biological fluorescence imaging,” Opt. Express 15(5), 2459–2467 (2007).
[Crossref] [PubMed]

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

M. C. Lang, J. Engelhardt, and S. W. Hell, “4Pi microscopy with linear fluorescence excitation,” Opt. Lett. 32(3), 259–261 (2007).
[Crossref] [PubMed]

T. Staudt, M. C. Lang, R. Medda, J. Engelhardt, and S. W. Hell, “2,2′-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy,” Microsc. Res. Tech. 70(1), 1–9 (2007).
[Crossref] [PubMed]

H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, “Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy,” Biophys. J. 87(6), 4146–4152 (2004).
[Crossref] [PubMed]

M. Dyba and S. W. Hell, “Focal spots of size lambda/23 open up far-field fluorescence microscopy at 33 nm axial resolution,” Phys. Rev. Lett. 88(16), 163901 (2002).
[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]

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.

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[Crossref] [PubMed]

Hofkens, J.

Hotta, J.

Hoyer, P.

F. Görlitz, P. Hoyer, H. Falk, L. Kastrup, J. Engelhardt, and S. W. Hell, “A STED microscope designed for routine biomedical applications,” Prog. Electromagnetics Res. 147, 57–68 (2014).
[Crossref]

Huang, B.

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319(5864), 810–813 (2008).
[Crossref] [PubMed]

Jakobs, S.

R. Schmidt, C. A. Wurm, A. Punge, A. Egner, S. Jakobs, and S. W. Hell, “Mitochondrial cristae revealed with focused light,” Nano Lett. 9(6), 2508–2510 (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. Methods 5(6), 539–544 (2008).
[Crossref] [PubMed]

H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, “Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy,” Biophys. J. 87(6), 4146–4152 (2004).
[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]

Johansson, G. A.

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A. 109(3), E135–E143 (2012).
[Crossref] [PubMed]

Kamps-Hughes, N.

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A. 109(3), E135–E143 (2012).
[Crossref] [PubMed]

Kanchanawong, P.

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[Crossref] [PubMed]

Kastrup, L.

F. Görlitz, P. Hoyer, H. Falk, L. Kastrup, J. Engelhardt, and S. W. Hell, “A STED microscope designed for routine biomedical applications,” Prog. Electromagnetics Res. 147, 57–68 (2014).
[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]

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]

Kirchhausen, T.

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349(6251), aab3500 (2015).
[Crossref] [PubMed]

Klar, T. A.

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

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]

Lang, M.

Lang, M. C.

M. C. Lang, T. Staudt, J. Engelhardt, and S. W. Hell, “4Pi microscopy with negligible sidelobes,” New J. Phys. 10(4), 043041 (2008).
[Crossref]

T. Staudt, M. C. Lang, R. Medda, J. Engelhardt, and S. W. Hell, “2,2′-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy,” Microsc. Res. Tech. 70(1), 1–9 (2007).
[Crossref] [PubMed]

M. C. Lang, J. Engelhardt, and S. W. Hell, “4Pi microscopy with linear fluorescence excitation,” Opt. Lett. 32(3), 259–261 (2007).
[Crossref] [PubMed]

Lang, T.

D. Aquino, A. Schönle, C. Geisler, C. V. Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, and A. Egner, “Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores,” Nat. Methods 8(4), 353–359 (2011).
[Crossref] [PubMed]

Li, D.

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349(6251), aab3500 (2015).
[Crossref] [PubMed]

Lippincott-Schwartz, J.

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[Crossref] [PubMed]

Macklin, J. J.

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A. 109(3), E135–E143 (2012).
[Crossref] [PubMed]

Manley, S.

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[Crossref] [PubMed]

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]

T. Staudt, M. C. Lang, R. Medda, J. Engelhardt, and S. W. Hell, “2,2′-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy,” Microsc. Res. Tech. 70(1), 1–9 (2007).
[Crossref] [PubMed]

Middendorff, C. V.

D. Aquino, A. Schönle, C. Geisler, C. V. Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, and A. Egner, “Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores,” Nat. Methods 8(4), 353–359 (2011).
[Crossref] [PubMed]

Milkie, D. E.

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349(6251), aab3500 (2015).
[Crossref] [PubMed]

Moses, B.

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349(6251), aab3500 (2015).
[Crossref] [PubMed]

Müller, T.

Muls, B.

Okamura, Y.

D. Aquino, A. Schönle, C. Geisler, C. V. Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, and A. Egner, “Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores,” Nat. Methods 8(4), 353–359 (2011).
[Crossref] [PubMed]

Pasham, M.

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349(6251), aab3500 (2015).
[Crossref] [PubMed]

Punge, A.

R. Schmidt, C. A. Wurm, A. Punge, A. Egner, S. Jakobs, and S. W. Hell, “Mitochondrial cristae revealed with focused light,” Nano Lett. 9(6), 2508–2510 (2009).
[Crossref] [PubMed]

Rego, E. H.

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A. 109(3), E135–E143 (2012).
[Crossref] [PubMed]

Reuss, M.

Richards, B.

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A Math. Phys. Sci. 253(1274), 358–379 (1959).
[Crossref]

Schmidt, R.

R. Schmidt, C. A. Wurm, A. Punge, A. Egner, S. Jakobs, and S. W. Hell, “Mitochondrial cristae revealed with focused light,” Nano Lett. 9(6), 2508–2510 (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. Methods 5(6), 539–544 (2008).
[Crossref] [PubMed]

Schönle, A.

D. Aquino, A. Schönle, C. Geisler, C. V. Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, and A. Egner, “Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores,” Nat. Methods 8(4), 353–359 (2011).
[Crossref] [PubMed]

Sedat, J. W.

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, “I5M: 3D widefield light microscopy with better than 100 nm axial resolution,” J. Microsc. 195(1), 10–16 (1999).
[Crossref] [PubMed]

Shao, L.

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349(6251), aab3500 (2015).
[Crossref] [PubMed]

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A. 109(3), E135–E143 (2012).
[Crossref] [PubMed]

Shtengel, G.

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[Crossref] [PubMed]

Sougrat, R.

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[Crossref] [PubMed]

Staudt, T.

M. C. Lang, T. Staudt, J. Engelhardt, and S. W. Hell, “4Pi microscopy with negligible sidelobes,” New J. Phys. 10(4), 043041 (2008).
[Crossref]

T. Staudt, M. C. Lang, R. Medda, J. Engelhardt, and S. W. Hell, “2,2′-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy,” Microsc. Res. Tech. 70(1), 1–9 (2007).
[Crossref] [PubMed]

Stelzer, E. H. K.

Storz, R.

H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, “Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy,” Biophys. J. 87(6), 4146–4152 (2004).
[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,” Science 319(5864), 810–813 (2008).
[Crossref] [PubMed]

Waterman, C. M.

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[Crossref] [PubMed]

Westphal, V.

Wichmann, J.

Wildanger, D.

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]

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]

Winoto, L.

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A. 109(3), E135–E143 (2012).
[Crossref] [PubMed]

Wolf, E.

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A Math. Phys. Sci. 253(1274), 358–379 (1959).
[Crossref]

E. Wolf, “Electromagnetic diffraction in optical systems I. An integral representation of the image field,” Proc. R. Soc. Lond. A Math. Phys. Sci. 253(1274), 349–357 (1959).
[Crossref]

Wurm, C. A.

D. Aquino, A. Schönle, C. Geisler, C. V. Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, and A. Egner, “Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores,” Nat. Methods 8(4), 353–359 (2011).
[Crossref] [PubMed]

R. Schmidt, C. A. Wurm, A. Punge, A. Egner, S. Jakobs, and S. W. Hell, “Mitochondrial cristae revealed with focused light,” Nano Lett. 9(6), 2508–2510 (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. Methods 5(6), 539–544 (2008).
[Crossref] [PubMed]

Xu, P.

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349(6251), aab3500 (2015).
[Crossref] [PubMed]

Zhang, M.

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349(6251), aab3500 (2015).
[Crossref] [PubMed]

Zhang, X.

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349(6251), aab3500 (2015).
[Crossref] [PubMed]

Zhuang, X.

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319(5864), 810–813 (2008).
[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]

Biophys. J. (1)

H. Gugel, J. Bewersdorf, S. Jakobs, J. Engelhardt, R. Storz, and S. W. Hell, “Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy,” Biophys. J. 87(6), 4146–4152 (2004).
[Crossref] [PubMed]

J. Microsc. (2)

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]

M. G. L. Gustafsson, D. A. Agard, and J. W. Sedat, “I5M: 3D widefield light microscopy with better than 100 nm axial resolution,” J. Microsc. 195(1), 10–16 (1999).
[Crossref] [PubMed]

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

Microsc. Res. Tech. (1)

T. Staudt, M. C. Lang, R. Medda, J. Engelhardt, and S. W. Hell, “2,2′-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy,” Microsc. Res. Tech. 70(1), 1–9 (2007).
[Crossref] [PubMed]

Nano Lett. (2)

R. Schmidt, C. A. Wurm, A. Punge, A. Egner, S. Jakobs, and S. W. Hell, “Mitochondrial cristae revealed with focused light,” Nano Lett. 9(6), 2508–2510 (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 (3)

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

R. Schmidt, C. A. Wurm, S. Jakobs, J. Engelhardt, A. Egner, and S. W. Hell, “Spherical nanosized focal spot unravels the interior of cells,” Nat. Methods 5(6), 539–544 (2008).
[Crossref] [PubMed]

D. Aquino, A. Schönle, C. Geisler, C. V. Middendorff, C. A. Wurm, Y. Okamura, T. Lang, S. W. Hell, and A. Egner, “Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores,” Nat. Methods 8(4), 353–359 (2011).
[Crossref] [PubMed]

New J. Phys. (1)

M. C. Lang, T. Staudt, J. Engelhardt, and S. W. Hell, “4Pi microscopy with negligible sidelobes,” New J. Phys. 10(4), 043041 (2008).
[Crossref]

Opt. Express (4)

Opt. Lett. (2)

Phys. Rev. Lett. (1)

M. Dyba and S. W. Hell, “Focal spots of size lambda/23 open up far-field fluorescence microscopy at 33 nm axial resolution,” Phys. Rev. Lett. 88(16), 163901 (2002).
[Crossref] [PubMed]

Proc. Natl. Acad. Sci. U.S.A. (4)

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[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]

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]

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A. 109(3), E135–E143 (2012).
[Crossref] [PubMed]

Proc. R. Soc. Lond. A Math. Phys. Sci. (2)

E. Wolf, “Electromagnetic diffraction in optical systems I. An integral representation of the image field,” Proc. R. Soc. Lond. A Math. Phys. Sci. 253(1274), 349–357 (1959).
[Crossref]

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. II. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A Math. Phys. Sci. 253(1274), 358–379 (1959).
[Crossref]

Prog. Electromagnetics Res. (1)

F. Görlitz, P. Hoyer, H. Falk, L. Kastrup, J. Engelhardt, and S. W. Hell, “A STED microscope designed for routine biomedical applications,” Prog. Electromagnetics Res. 147, 57–68 (2014).
[Crossref]

Science (3)

D. Li, L. Shao, B.-C. Chen, X. Zhang, M. Zhang, B. Moses, D. E. Milkie, J. R. Beach, J. A. Hammer, M. Pasham, T. Kirchhausen, M. A. Baird, M. W. Davidson, P. Xu, and E. Betzig, “Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics,” Science 349(6251), aab3500 (2015).
[Crossref] [PubMed]

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

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319(5864), 810–813 (2008).
[Crossref] [PubMed]

Other (3)

S. W. Hell, “Increasing the Resolution of Far-Field Fluorescence Microscopy by Point-Spread-Function Engineering,” in Topics in Fluorescence Spectroscopy 5: Nonlinear and Two-Photon-Induced Fluorescence, J. Lakowicz, ed. (Plenum, 1997), pp. 361–426.

R. Schmidt, “3D fluorescence microscopy with isotropic resolution on the nanoscale,” Ph.D. thesis (2008).

J. Bewersdorf, A. Egner, and S. W. Hell, “4Pi Microscopy,” in Handbook of Biological Confocal Microscopy, J. Pawley, ed. (Springer, 2006), pp. 561–570.

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Figures (6)

Fig. 1
Fig. 1 The segmented wave plate (SWP) or easySTED phase plate for isoSTED: (a) Polarization (orientation indicated by arrows) of the circularly polarized STED laser beam is modified differently for each segment of the SWP. Each segment acts as λ/2 wave plate for the STED wavelength. The lines in each segment indicate the orientation of the crystal’s optical axis. A subsequent λ/2 wave plate is necessary to generate z-polarized components in the resulting illumination pattern for axial STED. The necessity of a λ/2 wave plate can be avoided by flipping two segments of the SWP as shown on top. (b) Two mirror-symmetric, polarization-modified STED laser beams interfere in the common focus of two high-NA objective lenses. Interference renders a single point of zero intensity at the focal center. (c) Lateral confinement of the fluorescence intensity is given by x- and y- polarization components. Axial confinement is achieved by the z-polarized components. (d) Both lateral (dashed line) and axial (solid line) intensity profiles exhibit similar intensities in the vicinity of the focal center, which allows for isotropic STED. (e) Due to the chromatic selectivity of the SWP the excitation light remains almost a Gaussian beam and exhibits a 4Pi-characteristic interference pattern in the axial direction (shown on the right). Simulations of the 4Pi illumination patterns according to [27,28] with NA = 1.46, λexc = 635 nm and λSTED = 775 nm. Scale bars: 200 nm.
Fig. 2
Fig. 2 Working scheme of the single-beam isoSTED nanoscope. Excitation (EXC.) and STED lasers are coupled into a polarization-maintaining single mode fiber (SMF). A dichroic mirror (DM) reflects both laser lines. Light undergoes wavelength-selective polarization modification while passing the wave plates (WP). Scan lens (SL), beam scanner (BS) and tube lens (TL) allow for shifting of the focal excitation and STED beam profiles in both lateral directions of the focal plane. 4Pi optics is realized with a non-polarizing beam splitter cube (BSC) and two high-NA objective lenses (OL). The sample (S) is located in the focus of the OLs. Fluorescence light is detected by an avalanche photo diode (APD). A band pass filter (BP) filters out excitation and STED light, which does not reach the APD.
Fig. 3
Fig. 3 Design of the 4Pi nanoscopy platform. The optical paths were set up on an optical breadboard which was fixated vertically on an optical table. As a result, the sample is placed horizontally. The objective lens cage (dark grey) is independent of the interferometer. A front view of the interferometer and the objective lens cage is shown in (a). Black arrows indicate the degrees of freedom inside the cage. The dotted arrow shows the degree of freedom of the cage with respect to the beamsplitter and mirrors forming the 4Pi cavity. (b) Side view. (c) The objective lens cage can be removed (shown) and the interferometer can then be pre-aligned.
Fig. 4
Fig. 4 Image showing fluorescent microspheres in two axially distinct layers. In (a), an image along xz is shown. On the left side, the confocal image is shown as compared to the isoSTED image (right half). Resolution enhancement is substantiated over a wide axial range (at both the top and bottom coverglass surface). The distance between the cover glasses is ~8 μm. In (b) a xy and an yz image of a fluorescent microsphere are shown. Images were fitted with a two-dimensional Gaussian function (right). The histogram in (c) shows the FWHM of fit beads in a xy scan. Smallest detected FWHM were 20 nm, and most microspheres appeared with a size between 30 and 40 nm.
Fig. 5
Fig. 5 (a) 3D image of a Vero cell vimentin network stained with Abberior STAR635P. Features appear with a size of 60 nm; color codes the axial position as indicated. Overall image depth is 875 nm. The lower left corner shows a single 4Pi frame for comparison. (b) Vimentin network stained with Abberior STAR635P in a Vero cell at larger magnification. The color encodes the axial position.
Fig. 6
Fig. 6 Nuclear pore complexes in Vero cells: The protein nup153 immunolabeled with the dye Abberior Star Red. In (a), the xz cross-section (single-pixel 25 nm thickness) at the y position indicated by the white arrows is shown. In (b), the xy view with color- coded z position is shown, as compared to a 4Pi stack in the lower left corner of the image. Scale bar of 1 µm applies to both (a) and (b).

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