Abstract

Stimulated Emission Depletion (STED) nanoscopy enables multi-color fluorescence imaging at the nanometer scale. Its typical single-point scanning implementation can lead to long acquisition times. In order to unleash the full spatiotemporal resolution potential of STED nanoscopy, parallelized scanning is mandatory. Here we present a dual-color STED nanoscope utilizing two orthogonally crossed standing light waves as a fluorescence switch-off pattern, and providing a resolving power down to 30 nm. We demonstrate the imaging capabilities in a biological context for immunostained vimentin fibers in a circular field of view of 20 µm diameter at 2000-fold parallelization (i.e. 2000 “intensity minima”). The technical feasibility of massively parallelizing STED without significant compromises in resolution heralds video-rate STED nanoscopy of large fields of view, pending the availability of suitable high-speed detectors.

© 2015 Optical Society of America

Full Article  |  PDF Article
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2014 (3)

G. Lukinavičius, L. Reymond, E. D’Este, A. Masharina, F. Göttfert, H. Ta, A. Güther, M. Fournier, S. Rizzo, H. Waldmann, C. Blaukopf, C. Sommer, D. W. Gerlich, H.-D. Arndt, S. W. Hell, and K. Johnsson, “Fluorogenic probes for live-cell imaging of the cytoskeleton,” Nat. Methods 11(7), 731–733 (2014).
[Crossref] [PubMed]

B. Yang, F. Przybilla, M. Mestre, J.-B. Trebbia, and B. Lounis, “Large parallelization of STED nanoscopy using optical lattices,” Opt. Express 22(5), 5581–5589 (2014).
[Crossref] [PubMed]

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).

2013 (3)

G. Vicidomini, A. Schönle, H. Ta, K. Y. Han, G. Moneron, C. Eggeling, and S. W. Hell, “STED Nanoscopy with Time-Gated Detection: Theoretical and Experimental Aspects,” PLoS ONE 8(1), e54421 (2013).
[Crossref] [PubMed]

F. Göttfert, C. A. Wurm, V. Mueller, S. Berning, V. C. Cordes, A. Honigmann, and S. W. Hell, “Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution,” Biophys. J. 105(1), L01–L03 (2013).
[Crossref] [PubMed]

A. Chmyrov, J. Keller, T. Grotjohann, M. Ratz, E. d’Este, S. Jakobs, C. Eggeling, and S. W. Hell, “Nanoscopy with more than 100,000 ‘doughnuts’,” Nat. Methods 10(8), 737–740 (2013).
[Crossref] [PubMed]

2012 (3)

C. A. Wurm, K. Kolmakov, F. Göttfert, H. Ta, M. Bossi, H. Schill, S. Berning, S. Jakobs, G. Donnert, V. N. Belov, and S. W. Hell, “Novel red fluorophores with superior performance in STED microscopy,” Opt. Nanoscopy 1(1), 1–7 (2012).
[Crossref]

S. Berning, K. I. Willig, H. Steffens, P. Dibaj, and S. W. Hell, “Nanoscopy in a Living Mouse Brain,” Science 335(6068), 551 (2012).
[Crossref] [PubMed]

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. 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 (3)

J. Bückers, D. Wildanger, G. Vicidomini, L. Kastrup, and S. W. Hell, “Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses,” Opt. Express 19(4), 3130–3143 (2011).
[PubMed]

P. Bingen, M. Reuss, J. Engelhardt, and S. W. Hell, “Parallelized STED fluorescence nanoscopy,” Opt. Express 19(24), 23716–23726 (2011).
[Crossref] [PubMed]

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature 478(7368), 204–208 (2011).
[Crossref] [PubMed]

2009 (2)

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

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

2008 (3)

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[Crossref] [PubMed]

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement,” Science 320(5873), 246–249 (2008).
[Crossref] [PubMed]

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

2007 (1)

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]

2006 (4)

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]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (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,” Science 313(5793), 1642–1645 (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]

2005 (2)

M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U.S.A. 102(49), 17565–17569 (2005).
[Crossref] [PubMed]

M. G. 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 (1)

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

2002 (1)

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

2000 (2)

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

1994 (1)

1977 (1)

G. Porter, P. J. Sadkowski, and C. J. Tredwell, “Picosecond rotational diffusion in kinetic and steady state fluorescence spectroscopy,” Chem. Phys. Lett. 49(3), 416–420 (1977).
[Crossref]

Andrei, M. A.

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

Arndt, H.-D.

G. Lukinavičius, L. Reymond, E. D’Este, A. Masharina, F. Göttfert, H. Ta, A. Güther, M. Fournier, S. Rizzo, H. Waldmann, C. Blaukopf, C. Sommer, D. W. Gerlich, H.-D. Arndt, S. W. Hell, and K. Johnsson, “Fluorogenic probes for live-cell imaging of the cytoskeleton,” Nat. Methods 11(7), 731–733 (2014).
[Crossref] [PubMed]

Bates, M.

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

Belov, V. N.

C. A. Wurm, K. Kolmakov, F. Göttfert, H. Ta, M. Bossi, H. Schill, S. Berning, S. Jakobs, G. Donnert, V. N. Belov, and S. W. Hell, “Novel red fluorophores with superior performance in STED microscopy,” Opt. Nanoscopy 1(1), 1–7 (2012).
[Crossref]

Berning, S.

F. Göttfert, C. A. Wurm, V. Mueller, S. Berning, V. C. Cordes, A. Honigmann, and S. W. Hell, “Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution,” Biophys. J. 105(1), L01–L03 (2013).
[Crossref] [PubMed]

S. Berning, K. I. Willig, H. Steffens, P. Dibaj, and S. W. Hell, “Nanoscopy in a Living Mouse Brain,” Science 335(6068), 551 (2012).
[Crossref] [PubMed]

C. A. Wurm, K. Kolmakov, F. Göttfert, H. Ta, M. Bossi, H. Schill, S. Berning, S. Jakobs, G. Donnert, V. N. Belov, and S. W. Hell, “Novel red fluorophores with superior performance in STED microscopy,” Opt. Nanoscopy 1(1), 1–7 (2012).
[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,” Science 313(5793), 1642–1645 (2006).
[Crossref] [PubMed]

Bingen, P.

Blaukopf, C.

G. Lukinavičius, L. Reymond, E. D’Este, A. Masharina, F. Göttfert, H. Ta, A. Güther, M. Fournier, S. Rizzo, H. Waldmann, C. Blaukopf, C. Sommer, D. W. Gerlich, H.-D. Arndt, S. W. Hell, and K. Johnsson, “Fluorogenic probes for live-cell imaging of the cytoskeleton,” Nat. Methods 11(7), 731–733 (2014).
[Crossref] [PubMed]

Bock, H.

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature 478(7368), 204–208 (2011).
[Crossref] [PubMed]

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[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]

Bossi, M.

C. A. Wurm, K. Kolmakov, F. Göttfert, H. Ta, M. Bossi, H. Schill, S. Berning, S. Jakobs, G. Donnert, V. N. Belov, and S. W. Hell, “Novel red fluorophores with superior performance in STED microscopy,” Opt. Nanoscopy 1(1), 1–7 (2012).
[Crossref]

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[Crossref] [PubMed]

Bückers, J.

Charbon, E.

Y. Maruyama and E. Charbon, “A time-gated 128x128 CMOS SPAD array for on-chip fluorescence detection,” in Proc. Intl. Image Sensor Workshop (IISW) (2011), pp. 270–273.

Chmyrov, A.

A. Chmyrov, J. Keller, T. Grotjohann, M. Ratz, E. d’Este, S. Jakobs, C. Eggeling, and S. W. Hell, “Nanoscopy with more than 100,000 ‘doughnuts’,” Nat. Methods 10(8), 737–740 (2013).
[Crossref] [PubMed]

Cordes, V. C.

F. Göttfert, C. A. Wurm, V. Mueller, S. Berning, V. C. Cordes, A. Honigmann, and S. W. Hell, “Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution,” Biophys. J. 105(1), L01–L03 (2013).
[Crossref] [PubMed]

D’Este, E.

G. Lukinavičius, L. Reymond, E. D’Este, A. Masharina, F. Göttfert, H. Ta, A. Güther, M. Fournier, S. Rizzo, H. Waldmann, C. Blaukopf, C. Sommer, D. W. Gerlich, H.-D. Arndt, S. W. Hell, and K. Johnsson, “Fluorogenic probes for live-cell imaging of the cytoskeleton,” Nat. Methods 11(7), 731–733 (2014).
[Crossref] [PubMed]

A. Chmyrov, J. Keller, T. Grotjohann, M. Ratz, E. d’Este, S. Jakobs, C. Eggeling, and S. W. Hell, “Nanoscopy with more than 100,000 ‘doughnuts’,” Nat. Methods 10(8), 737–740 (2013).
[Crossref] [PubMed]

Davidson, M. W.

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. 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]

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]

Dibaj, P.

S. Berning, K. I. Willig, H. Steffens, P. Dibaj, and S. W. Hell, “Nanoscopy in a Living Mouse Brain,” Science 335(6068), 551 (2012).
[Crossref] [PubMed]

Donnert, G.

C. A. Wurm, K. Kolmakov, F. Göttfert, H. Ta, M. Bossi, H. Schill, S. Berning, S. Jakobs, G. Donnert, V. N. Belov, and S. W. Hell, “Novel red fluorophores with superior performance in STED microscopy,” Opt. Nanoscopy 1(1), 1–7 (2012).
[Crossref]

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

Dyba, M.

M. Dyba and S. W. Hell, “Focal spots of size λ/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]

Eggeling, C.

A. Chmyrov, J. Keller, T. Grotjohann, M. Ratz, E. d’Este, S. Jakobs, C. Eggeling, and S. W. Hell, “Nanoscopy with more than 100,000 ‘doughnuts’,” Nat. Methods 10(8), 737–740 (2013).
[Crossref] [PubMed]

G. Vicidomini, A. Schönle, H. Ta, K. Y. Han, G. Moneron, C. Eggeling, and S. W. Hell, “STED Nanoscopy with Time-Gated Detection: Theoretical and Experimental Aspects,” PLoS ONE 8(1), e54421 (2013).
[Crossref] [PubMed]

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature 478(7368), 204–208 (2011).
[Crossref] [PubMed]

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

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[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]

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]

M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U.S.A. 102(49), 17565–17569 (2005).
[Crossref] [PubMed]

Egner, A.

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

Engelhardt, J.

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).

P. Bingen, M. Reuss, J. Engelhardt, and S. W. Hell, “Parallelized STED fluorescence nanoscopy,” Opt. Express 19(24), 23716–23726 (2011).
[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).

Fölling, J.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[Crossref] [PubMed]

Fournier, M.

G. Lukinavičius, L. Reymond, E. D’Este, A. Masharina, F. Göttfert, H. Ta, A. Güther, M. Fournier, S. Rizzo, H. Waldmann, C. Blaukopf, C. Sommer, D. W. Gerlich, H.-D. Arndt, S. W. Hell, and K. Johnsson, “Fluorogenic probes for live-cell imaging of the cytoskeleton,” Nat. Methods 11(7), 731–733 (2014).
[Crossref] [PubMed]

Gerlich, D. W.

G. Lukinavičius, L. Reymond, E. D’Este, A. Masharina, F. Göttfert, H. Ta, A. Güther, M. Fournier, S. Rizzo, H. Waldmann, C. Blaukopf, C. Sommer, D. W. Gerlich, H.-D. Arndt, S. W. Hell, and K. Johnsson, “Fluorogenic probes for live-cell imaging of the cytoskeleton,” Nat. Methods 11(7), 731–733 (2014).
[Crossref] [PubMed]

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]

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).

Göttfert, F.

G. Lukinavičius, L. Reymond, E. D’Este, A. Masharina, F. Göttfert, H. Ta, A. Güther, M. Fournier, S. Rizzo, H. Waldmann, C. Blaukopf, C. Sommer, D. W. Gerlich, H.-D. Arndt, S. W. Hell, and K. Johnsson, “Fluorogenic probes for live-cell imaging of the cytoskeleton,” Nat. Methods 11(7), 731–733 (2014).
[Crossref] [PubMed]

F. Göttfert, C. A. Wurm, V. Mueller, S. Berning, V. C. Cordes, A. Honigmann, and S. W. Hell, “Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution,” Biophys. J. 105(1), L01–L03 (2013).
[Crossref] [PubMed]

C. A. Wurm, K. Kolmakov, F. Göttfert, H. Ta, M. Bossi, H. Schill, S. Berning, S. Jakobs, G. Donnert, V. N. Belov, and S. W. Hell, “Novel red fluorophores with superior performance in STED microscopy,” Opt. Nanoscopy 1(1), 1–7 (2012).
[Crossref]

Grotjohann, T.

A. Chmyrov, J. Keller, T. Grotjohann, M. Ratz, E. d’Este, S. Jakobs, C. Eggeling, and S. W. Hell, “Nanoscopy with more than 100,000 ‘doughnuts’,” Nat. Methods 10(8), 737–740 (2013).
[Crossref] [PubMed]

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature 478(7368), 204–208 (2011).
[Crossref] [PubMed]

Gustafsson, M. G.

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

Gustafsson, M. G. L.

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. 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]

Güther, A.

G. Lukinavičius, L. Reymond, E. D’Este, A. Masharina, F. Göttfert, H. Ta, A. Güther, M. Fournier, S. Rizzo, H. Waldmann, C. Blaukopf, C. Sommer, D. W. Gerlich, H.-D. Arndt, S. W. Hell, and K. Johnsson, “Fluorogenic probes for live-cell imaging of the cytoskeleton,” Nat. Methods 11(7), 731–733 (2014).
[Crossref] [PubMed]

Han, K. Y.

G. Vicidomini, A. Schönle, H. Ta, K. Y. Han, G. Moneron, C. Eggeling, and S. W. Hell, “STED Nanoscopy with Time-Gated Detection: Theoretical and Experimental Aspects,” PLoS ONE 8(1), e54421 (2013).
[Crossref] [PubMed]

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

Harke, B.

Hein, B.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[Crossref] [PubMed]

Hell, S. W.

G. Lukinavičius, L. Reymond, E. D’Este, A. Masharina, F. Göttfert, H. Ta, A. Güther, M. Fournier, S. Rizzo, H. Waldmann, C. Blaukopf, C. Sommer, D. W. Gerlich, H.-D. Arndt, S. W. Hell, and K. Johnsson, “Fluorogenic probes for live-cell imaging of the cytoskeleton,” Nat. Methods 11(7), 731–733 (2014).
[Crossref] [PubMed]

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).

G. Vicidomini, A. Schönle, H. Ta, K. Y. Han, G. Moneron, C. Eggeling, and S. W. Hell, “STED Nanoscopy with Time-Gated Detection: Theoretical and Experimental Aspects,” PLoS ONE 8(1), e54421 (2013).
[Crossref] [PubMed]

A. Chmyrov, J. Keller, T. Grotjohann, M. Ratz, E. d’Este, S. Jakobs, C. Eggeling, and S. W. Hell, “Nanoscopy with more than 100,000 ‘doughnuts’,” Nat. Methods 10(8), 737–740 (2013).
[Crossref] [PubMed]

F. Göttfert, C. A. Wurm, V. Mueller, S. Berning, V. C. Cordes, A. Honigmann, and S. W. Hell, “Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution,” Biophys. J. 105(1), L01–L03 (2013).
[Crossref] [PubMed]

S. Berning, K. I. Willig, H. Steffens, P. Dibaj, and S. W. Hell, “Nanoscopy in a Living Mouse Brain,” Science 335(6068), 551 (2012).
[Crossref] [PubMed]

C. A. Wurm, K. Kolmakov, F. Göttfert, H. Ta, M. Bossi, H. Schill, S. Berning, S. Jakobs, G. Donnert, V. N. Belov, and S. W. Hell, “Novel red fluorophores with superior performance in STED microscopy,” Opt. Nanoscopy 1(1), 1–7 (2012).
[Crossref]

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature 478(7368), 204–208 (2011).
[Crossref] [PubMed]

P. Bingen, M. Reuss, J. Engelhardt, and S. W. Hell, “Parallelized STED fluorescence nanoscopy,” Opt. Express 19(24), 23716–23726 (2011).
[Crossref] [PubMed]

J. Bückers, D. Wildanger, G. Vicidomini, L. Kastrup, and S. W. Hell, “Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses,” Opt. Express 19(4), 3130–3143 (2011).
[PubMed]

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

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

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[Crossref] [PubMed]

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement,” Science 320(5873), 246–249 (2008).
[Crossref] [PubMed]

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

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]

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]

M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U.S.A. 102(49), 17565–17569 (2005).
[Crossref] [PubMed]

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

M. Dyba and S. W. Hell, “Focal spots of size λ/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 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. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U.S.A. 102(49), 17565–17569 (2005).
[Crossref] [PubMed]

Honigmann, A.

F. Göttfert, C. A. Wurm, V. Mueller, S. Berning, V. C. Cordes, A. Honigmann, and S. W. Hell, “Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution,” Biophys. J. 105(1), L01–L03 (2013).
[Crossref] [PubMed]

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).

Irvine, S. E.

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

Jahn, R.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement,” Science 320(5873), 246–249 (2008).
[Crossref] [PubMed]

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.

A. Chmyrov, J. Keller, T. Grotjohann, M. Ratz, E. d’Este, S. Jakobs, C. Eggeling, and S. W. Hell, “Nanoscopy with more than 100,000 ‘doughnuts’,” Nat. Methods 10(8), 737–740 (2013).
[Crossref] [PubMed]

C. A. Wurm, K. Kolmakov, F. Göttfert, H. Ta, M. Bossi, H. Schill, S. Berning, S. Jakobs, G. Donnert, V. N. Belov, and S. W. Hell, “Novel red fluorophores with superior performance in STED microscopy,” Opt. Nanoscopy 1(1), 1–7 (2012).
[Crossref]

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature 478(7368), 204–208 (2011).
[Crossref] [PubMed]

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[Crossref] [PubMed]

M. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U.S.A. 102(49), 17565–17569 (2005).
[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. L. 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]

Johnsson, K.

G. Lukinavičius, L. Reymond, E. D’Este, A. Masharina, F. Göttfert, H. Ta, A. Güther, M. Fournier, S. Rizzo, H. Waldmann, C. Blaukopf, C. Sommer, D. W. Gerlich, H.-D. Arndt, S. W. Hell, and K. Johnsson, “Fluorogenic probes for live-cell imaging of the cytoskeleton,” Nat. Methods 11(7), 731–733 (2014).
[Crossref] [PubMed]

Kamin, D.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement,” Science 320(5873), 246–249 (2008).
[Crossref] [PubMed]

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. L. 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]

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).

J. Bückers, D. Wildanger, G. Vicidomini, L. Kastrup, and S. W. Hell, “Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses,” Opt. Express 19(4), 3130–3143 (2011).
[PubMed]

Keller, J.

A. Chmyrov, J. Keller, T. Grotjohann, M. Ratz, E. d’Este, S. Jakobs, C. Eggeling, and S. W. Hell, “Nanoscopy with more than 100,000 ‘doughnuts’,” Nat. Methods 10(8), 737–740 (2013).
[Crossref] [PubMed]

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

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.

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]

Kolmakov, K.

C. A. Wurm, K. Kolmakov, F. Göttfert, H. Ta, M. Bossi, H. Schill, S. Berning, S. Jakobs, G. Donnert, V. N. Belov, and S. W. Hell, “Novel red fluorophores with superior performance in STED microscopy,” Opt. Nanoscopy 1(1), 1–7 (2012).
[Crossref]

Lauterbach, M. A.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement,” Science 320(5873), 246–249 (2008).
[Crossref] [PubMed]

Lavoie-Cardinal, F.

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature 478(7368), 204–208 (2011).
[Crossref] [PubMed]

Leutenegger, M.

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature 478(7368), 204–208 (2011).
[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,” 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]

Lounis, B.

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]

Lukinavicius, G.

G. Lukinavičius, L. Reymond, E. D’Este, A. Masharina, F. Göttfert, H. Ta, A. Güther, M. Fournier, S. Rizzo, H. Waldmann, C. Blaukopf, C. Sommer, D. W. Gerlich, H.-D. Arndt, S. W. Hell, and K. Johnsson, “Fluorogenic probes for live-cell imaging of the cytoskeleton,” Nat. Methods 11(7), 731–733 (2014).
[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. L. 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]

Maruyama, Y.

Y. Maruyama and E. Charbon, “A time-gated 128x128 CMOS SPAD array for on-chip fluorescence detection,” in Proc. Intl. Image Sensor Workshop (IISW) (2011), pp. 270–273.

Masharina, A.

G. Lukinavičius, L. Reymond, E. D’Este, A. Masharina, F. Göttfert, H. Ta, A. Güther, M. Fournier, S. Rizzo, H. Waldmann, C. Blaukopf, C. Sommer, D. W. Gerlich, H.-D. Arndt, S. W. Hell, and K. Johnsson, “Fluorogenic probes for live-cell imaging of the cytoskeleton,” Nat. Methods 11(7), 731–733 (2014).
[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]

Medda, R.

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[Crossref] [PubMed]

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

Mestre, M.

Moneron, G.

G. Vicidomini, A. Schönle, H. Ta, K. Y. Han, G. Moneron, C. Eggeling, and S. W. Hell, “STED Nanoscopy with Time-Gated Detection: Theoretical and Experimental Aspects,” PLoS ONE 8(1), e54421 (2013).
[Crossref] [PubMed]

Mueller, V.

F. Göttfert, C. A. Wurm, V. Mueller, S. Berning, V. C. Cordes, A. Honigmann, and S. W. Hell, “Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution,” Biophys. J. 105(1), L01–L03 (2013).
[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,” 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]

Porter, G.

G. Porter, P. J. Sadkowski, and C. J. Tredwell, “Picosecond rotational diffusion in kinetic and steady state fluorescence spectroscopy,” Chem. Phys. Lett. 49(3), 416–420 (1977).
[Crossref]

Przybilla, F.

Ratz, M.

A. Chmyrov, J. Keller, T. Grotjohann, M. Ratz, E. d’Este, S. Jakobs, C. Eggeling, and S. W. Hell, “Nanoscopy with more than 100,000 ‘doughnuts’,” Nat. Methods 10(8), 737–740 (2013).
[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. L. 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.

Reymond, L.

G. Lukinavičius, L. Reymond, E. D’Este, A. Masharina, F. Göttfert, H. Ta, A. Güther, M. Fournier, S. Rizzo, H. Waldmann, C. Blaukopf, C. Sommer, D. W. Gerlich, H.-D. Arndt, S. W. Hell, and K. Johnsson, “Fluorogenic probes for live-cell imaging of the cytoskeleton,” Nat. Methods 11(7), 731–733 (2014).
[Crossref] [PubMed]

Rittweger, E.

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

Rizzo, S.

G. Lukinavičius, L. Reymond, E. D’Este, A. Masharina, F. Göttfert, H. Ta, A. Güther, M. Fournier, S. Rizzo, H. Waldmann, C. Blaukopf, C. Sommer, D. W. Gerlich, H.-D. Arndt, S. W. Hell, and K. Johnsson, “Fluorogenic probes for live-cell imaging of the cytoskeleton,” Nat. Methods 11(7), 731–733 (2014).
[Crossref] [PubMed]

Rizzoli, S. O.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement,” Science 320(5873), 246–249 (2008).
[Crossref] [PubMed]

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]

Rust, M. J.

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

Sadkowski, P. J.

G. Porter, P. J. Sadkowski, and C. J. Tredwell, “Picosecond rotational diffusion in kinetic and steady state fluorescence spectroscopy,” Chem. Phys. Lett. 49(3), 416–420 (1977).
[Crossref]

Schill, H.

C. A. Wurm, K. Kolmakov, F. Göttfert, H. Ta, M. Bossi, H. Schill, S. Berning, S. Jakobs, G. Donnert, V. N. Belov, and S. W. Hell, “Novel red fluorophores with superior performance in STED microscopy,” Opt. Nanoscopy 1(1), 1–7 (2012).
[Crossref]

Schönle, A.

G. Vicidomini, A. Schönle, H. Ta, K. Y. Han, G. Moneron, C. Eggeling, and S. W. Hell, “STED Nanoscopy with Time-Gated Detection: Theoretical and Experimental Aspects,” PLoS ONE 8(1), e54421 (2013).
[Crossref] [PubMed]

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

Shao, L.

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. 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]

Sommer, C.

G. Lukinavičius, L. Reymond, E. D’Este, A. Masharina, F. Göttfert, H. Ta, A. Güther, M. Fournier, S. Rizzo, H. Waldmann, C. Blaukopf, C. Sommer, D. W. Gerlich, H.-D. Arndt, S. W. Hell, and K. Johnsson, “Fluorogenic probes for live-cell imaging of the cytoskeleton,” Nat. Methods 11(7), 731–733 (2014).
[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]

Steffens, H.

S. Berning, K. I. Willig, H. Steffens, P. Dibaj, and S. W. Hell, “Nanoscopy in a Living Mouse Brain,” Science 335(6068), 551 (2012).
[Crossref] [PubMed]

Ta, H.

G. Lukinavičius, L. Reymond, E. D’Este, A. Masharina, F. Göttfert, H. Ta, A. Güther, M. Fournier, S. Rizzo, H. Waldmann, C. Blaukopf, C. Sommer, D. W. Gerlich, H.-D. Arndt, S. W. Hell, and K. Johnsson, “Fluorogenic probes for live-cell imaging of the cytoskeleton,” Nat. Methods 11(7), 731–733 (2014).
[Crossref] [PubMed]

G. Vicidomini, A. Schönle, H. Ta, K. Y. Han, G. Moneron, C. Eggeling, and S. W. Hell, “STED Nanoscopy with Time-Gated Detection: Theoretical and Experimental Aspects,” PLoS ONE 8(1), e54421 (2013).
[Crossref] [PubMed]

C. A. Wurm, K. Kolmakov, F. Göttfert, H. Ta, M. Bossi, H. Schill, S. Berning, S. Jakobs, G. Donnert, V. N. Belov, and S. W. Hell, “Novel red fluorophores with superior performance in STED microscopy,” Opt. Nanoscopy 1(1), 1–7 (2012).
[Crossref]

Testa, I.

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature 478(7368), 204–208 (2011).
[Crossref] [PubMed]

Trebbia, J.-B.

Tredwell, C. J.

G. Porter, P. J. Sadkowski, and C. J. Tredwell, “Picosecond rotational diffusion in kinetic and steady state fluorescence spectroscopy,” Chem. Phys. Lett. 49(3), 416–420 (1977).
[Crossref]

Ullal, C. K.

Urban, N. T.

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature 478(7368), 204–208 (2011).
[Crossref] [PubMed]

Vicidomini, G.

G. Vicidomini, A. Schönle, H. Ta, K. Y. Han, G. Moneron, C. Eggeling, and S. W. Hell, “STED Nanoscopy with Time-Gated Detection: Theoretical and Experimental Aspects,” PLoS ONE 8(1), e54421 (2013).
[Crossref] [PubMed]

J. Bückers, D. Wildanger, G. Vicidomini, L. Kastrup, and S. W. Hell, “Simultaneous multi-lifetime multi-color STED imaging for colocalization analyses,” Opt. Express 19(4), 3130–3143 (2011).
[PubMed]

Waldmann, H.

G. Lukinavičius, L. Reymond, E. D’Este, A. Masharina, F. Göttfert, H. Ta, A. Güther, M. Fournier, S. Rizzo, H. Waldmann, C. Blaukopf, C. Sommer, D. W. Gerlich, H.-D. Arndt, S. W. Hell, and K. Johnsson, “Fluorogenic probes for live-cell imaging of the cytoskeleton,” Nat. Methods 11(7), 731–733 (2014).
[Crossref] [PubMed]

Westphal, V.

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement,” Science 320(5873), 246–249 (2008).
[Crossref] [PubMed]

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

Wichmann, J.

Wildanger, D.

Willig, K. I.

S. Berning, K. I. Willig, H. Steffens, P. Dibaj, and S. W. Hell, “Nanoscopy in a Living Mouse Brain,” Science 335(6068), 551 (2012).
[Crossref] [PubMed]

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature 478(7368), 204–208 (2011).
[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. L. 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]

Wurm, C. A.

F. Göttfert, C. A. Wurm, V. Mueller, S. Berning, V. C. Cordes, A. Honigmann, and S. W. Hell, “Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution,” Biophys. J. 105(1), L01–L03 (2013).
[Crossref] [PubMed]

C. A. Wurm, K. Kolmakov, F. Göttfert, H. Ta, M. Bossi, H. Schill, S. Berning, S. Jakobs, G. Donnert, V. N. Belov, and S. W. Hell, “Novel red fluorophores with superior performance in STED microscopy,” Opt. Nanoscopy 1(1), 1–7 (2012).
[Crossref]

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[Crossref] [PubMed]

Yang, B.

Zhuang, X.

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

Biophys. J. (2)

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]

F. Göttfert, C. A. Wurm, V. Mueller, S. Berning, V. C. Cordes, A. Honigmann, and S. W. Hell, “Coaligned Dual-Channel STED Nanoscopy and Molecular Diffusion Analysis at 20 nm Resolution,” Biophys. J. 105(1), L01–L03 (2013).
[Crossref] [PubMed]

Chem. Phys. Lett. (1)

G. Porter, P. J. Sadkowski, and C. J. Tredwell, “Picosecond rotational diffusion in kinetic and steady state fluorescence spectroscopy,” Chem. Phys. Lett. 49(3), 416–420 (1977).
[Crossref]

J. Microsc. (1)

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

Nat. Biotechnol. (1)

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

Nat. Methods (6)

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

J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling, and S. W. Hell, “Fluorescence nanoscopy by ground-state depletion and single-molecule return,” Nat. Methods 5(11), 943–945 (2008).
[Crossref] [PubMed]

G. Lukinavičius, L. Reymond, E. D’Este, A. Masharina, F. Göttfert, H. Ta, A. Güther, M. Fournier, S. Rizzo, H. Waldmann, C. Blaukopf, C. Sommer, D. W. Gerlich, H.-D. Arndt, S. W. Hell, and K. Johnsson, “Fluorogenic probes for live-cell imaging of the cytoskeleton,” Nat. Methods 11(7), 731–733 (2014).
[Crossref] [PubMed]

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

A. Chmyrov, J. Keller, T. Grotjohann, M. Ratz, E. d’Este, S. Jakobs, C. Eggeling, and S. W. Hell, “Nanoscopy with more than 100,000 ‘doughnuts’,” Nat. Methods 10(8), 737–740 (2013).
[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]

Nat. Photonics (1)

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

Nature (1)

T. Grotjohann, I. Testa, M. Leutenegger, H. Bock, N. T. Urban, F. Lavoie-Cardinal, K. I. Willig, C. Eggeling, S. Jakobs, and S. W. Hell, “Diffraction-unlimited all-optical imaging and writing with a photochromic GFP,” Nature 478(7368), 204–208 (2011).
[Crossref] [PubMed]

Opt. Express (4)

Opt. Lett. (1)

Opt. Nanoscopy (1)

C. A. Wurm, K. Kolmakov, F. Göttfert, H. Ta, M. Bossi, H. Schill, S. Berning, S. Jakobs, G. Donnert, V. N. Belov, and S. W. Hell, “Novel red fluorophores with superior performance in STED microscopy,” Opt. Nanoscopy 1(1), 1–7 (2012).
[Crossref]

Phys. Rev. Lett. (1)

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

PLoS ONE (1)

G. Vicidomini, A. Schönle, H. Ta, K. Y. Han, G. Moneron, C. Eggeling, and S. W. Hell, “STED Nanoscopy with Time-Gated Detection: Theoretical and Experimental Aspects,” PLoS ONE 8(1), e54421 (2013).
[Crossref] [PubMed]

Proc. Natl. Acad. Sci. U.S.A. (5)

M. G. 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. L. 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. 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, 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. Hofmann, C. Eggeling, S. Jakobs, and S. W. Hell, “Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins,” Proc. Natl. Acad. Sci. U.S.A. 102(49), 17565–17569 (2005).
[Crossref] [PubMed]

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).

Science (3)

S. Berning, K. I. Willig, H. Steffens, P. Dibaj, and S. W. Hell, “Nanoscopy in a Living Mouse Brain,” Science 335(6068), 551 (2012).
[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]

V. Westphal, S. O. Rizzoli, M. A. Lauterbach, D. Kamin, R. Jahn, and S. W. Hell, “Video-Rate Far-Field Optical Nanoscopy Dissects Synaptic Vesicle Movement,” Science 320(5873), 246–249 (2008).
[Crossref] [PubMed]

Other (3)

RESOLFT: Reversible Saturable/Switchable Optically Linear (Fluorescence) Transitions.

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

Y. Maruyama and E. Charbon, “A time-gated 128x128 CMOS SPAD array for on-chip fluorescence detection,” in Proc. Intl. Image Sensor Workshop (IISW) (2011), pp. 270–273.

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Figures (5)

Fig. 1
Fig. 1

Widefield excitation (green) and patterned off-switching (red) of fluorophores realize the STED concept on a massively parallelized scale. (a) STED off-switching pattern produced as standing wave by interference of crossed beams. (b) Confined fluorescence signals (yellow) stemming from the targeted sample regions are imaged onto the EMCCD camera and (c) assigned to their corresponding pixels in the high-resolution image. The STED pattern is scanned across the sample on a slight diagonal path via piezo-controlled optical path length changes (“piezo scan”) in the arms of two Michelson interferometers. The second orthogonal interferometer combination is not shown for simplicity. (d) Details of STED beam path before coupling into the microscope, including both interferometer arrangements. (e) Example of raw data detail, scale bar is 2 µm (in sample plane). PBS: polarizing beam splitter, DC: dichroic beamsplitter, TL: tube lens, OL: objective lens, λ/2: half-wave plate, λ/4: quarter-wave plate, SP: short pass filter, EMCCD: electron-multiplying charge-coupled device, EXC: Excitation.

Fig. 2
Fig. 2

Resolution quantification. (a-c) Parallelized STED vs. widefield image of 20 nm Crimson beads with magnified inset. (d) The bead images were fit with a 2D Gaussian model function and full width at half maximum (FWHM) fit values are displayed color-coded in a scatter plot (corresponding to full region of (a)). (e) Histogram of the FWHM fit values.

Fig. 3
Fig. 3

(a) 2000-fold parallelized dual-color STED imaging of 200 nm fluorescent beads via excitation multiplexing: Crimson beads (intensity scales from black to green color) excited at 600 nm; Dark Red beads (black to red color) at 650 nm. Considerable cross-excitation at 650 nm is largely removed via linear unmixing. Note the increased image brightness near the periphery as a consequence of lower resolution due to fall-off of the STED intensity envelope. (b,c) Magnified view of boxed region in (a).

Fig. 4
Fig. 4

2500-fold parallelized STED imaging of biological structures. (a) STAR 635P-stained vimentin fibers. Magnified inset regions (b,c) and intensity profile plots (d,e) at dashed line show widefield (top) and STED recordings (bottom) in comparison. STED profile data is overlaid with a Gaussian fit model (red line).

Fig. 5
Fig. 5

Resolution in coordinate-targeted nanoscopy. (a) s·h(x) for doughnut STED (dSTED) and parallelized STED (pSTED) for the case θ equal to the aperture angle α. Same curvature near the zero (i.e. same quadratic approximation) yields the same resolution, cf. close-up shown in (b). The resolution enhancement (given by the FWHM) scales with 1/√(s). (c) “ON”-state probability pon = pexc·(f(s·h(x)). (d) Exponential decay pON = f of population of the excited fluorescent state versus saturation parameter s. pON is normalized to 1.

Equations (7)

Equations on this page are rendered with MathJax. Learn more.

p O N ( x ) = p e x c ( x 0 ) p e x c ( 0 ) p S T E D ( x 0 ) = ! 1 2
exp [ l n ( 2 ) ( 1 ( w e x c / 2 ) 2 + s e ( w d / 2 ) 2 ) x 0 2 ] = ! 1 2
( 1 + s e 1.86 2 ) ( 2 x 0 ) 2 w f l 2 = 1
d = λ f l 2 N A 1 1 + 0.8 s
exp [ ln ( 2 ) ( 1 ( w θ / 4 ) 2 + s ( π w θ ) 2 ) x 0 2 ] = ! 1 2 ( 4 + s ( π 2 ) 2 ) ( 2 x 0 ) 2 1 w θ 2 w α , S T E D 2 w α , S T E D 2 = ! 1 d = λ S T E D 2 N A 1 4 + ( π / 2 ) 2 s sin θ sin α = λ f l 2 N A 1 3.25 + 2 s sin θ sin α
s d h d = ! s p h p s d s p = I d I p = ( π / w θ ) 2 e ( w d / 2 ) 2 = π 2 e ( 1.68 2 ) 2 = 2.56 sin 2 θ sin 2 α
P d P p = I d I p π e ( w d / 2 ) 2 w θ 2 = π 3 ( 1.68 2 ) 4 ( sin θ sin α ) 4 = 15.4 sin 4 θ sin 4 α

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