Abstract

Stimulated emission depletion (STED) fluorescence microscopy squeezes an excited spot well below the wavelength scale using a doughnut-shaped depletion beam. To generate a doughnut, a scale-free vortex phase modulation (2D-STED) is often used because it provides maximal transverse confinement and radial-aberration immunity (RAI) to the central dip. However, RAI also means blindness to a defocus term, making the axial origin of fluorescence photons uncertain within the wavelength scale provided by the confocal detection pinhole. Here, to reduce the uncertainty, we perturb the 2D-STED phase mask so as to change the sign of the axial concavity near focus, creating a dilated dip. By providing laser depletion power, the dip can be compressed back in three dimensions to retrieve lateral resolution, now at a significantly higher contrast. We test this coherent-hybrid STED (CH-STED) mode in x-y imaging of complex biological structures, such as the dividing cell. The proposed strategy creates an orthogonal direction in the STED parametric space that uniquely allows independent tuning of resolution and contrast using a single depletion beam in a conventional (circular polarization-based) STED setup.

© 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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2018 (3)

T. Kaldewey, A. V. Kuhlmann, S. R. Valentin, A. Ludwig, A. D. Wieck, and R. J. Warburton, “Far-field nanoscopy on a semiconductor quantum dot via a rapid-adiabatic-passage-based switch,” Nat. Photonics 12(2), 68–72 (2018).
[Crossref]

J. Heine, C. A. Wurm, J. Keller-Findeisen, A. Schönle, B. Harke, M. Reuss, F. R. Winter, and G. Donnert, “Three dimensional live-cell STED microscopy at increased depth using a water immersion objective,” Rev. Sci. Instrum. 89(5), 053701 (2018).
[Crossref] [PubMed]

L. Wang, B. Chen, W. Yan, Z. Yang, X. Peng, D. Lin, X. Weng, T. Ye, and J. Qu, “Resolution improvement in STED super-resolution microscopy at low power using a phasor plot approach,” Nanoscale 10(34), 16252–16260 (2018).
[Crossref] [PubMed]

2017 (7)

J. Heine, M. Reuss, B. Harke, E. D’Este, S. J. Sahl, and S. W. Hell, “Adaptive-illumination STED nanoscopy,” Proc. Natl. Acad. Sci. U.S.A. 114(37), 9797–9802 (2017).
[Crossref] [PubMed]

F. Göttfert, T. Pleiner, J. Heine, V. Westphal, D. Görlich, S. J. Sahl, and S. W. Hell, “Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent,” Proc. Natl. Acad. Sci. U.S.A. 114(9), 2125–2130 (2017).
[Crossref] [PubMed]

K. Sozanski, E. Sisamakis, X. Zhang, and R. Holyst, “Quantitative fluorescence correlation spectroscopy in three-dimensional systems under stimulated emission depletion conditions,” Optica 4(8), 982–988 (2017).
[Crossref]

P. Gao, B. Prunsche, L. Zhou, K. Nienhaus, and G. U. Nienhaus, “Background suppression in fluorescence nanoscopy with stimulated emission double depletion,” Nat. Photonics 11(3), 163–169 (2017).
[Crossref]

F. Balzarotti, Y. Eilers, K. C. Gwosch, A. H. Gynnå, V. Westphal, F. D. Stefani, J. Elf, and S. W. Hell, “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes,” Science 355(6325), 606–612 (2017).
[Crossref] [PubMed]

J. Antonello, D. Burke, and M. J. Booth, “Aberrations in stimulated emission depletion (STED) microscopy,” Opt. Commun. 404, 203–209 (2017).
[Crossref] [PubMed]

B. Wang, J. Shi, T. Zhang, X. Xu, Y. Cao, and X. Li, “Improved lateral resolution with an annular vortex depletion beam in STED microscopy,” Opt. Lett. 42(23), 4885–4888 (2017).
[Crossref] [PubMed]

2016 (3)

J. Antonello, E. B. Kromann, D. Burke, J. Bewersdorf, and M. J. Booth, “Coma aberrations in combined two- and three-dimensional STED nanoscopy,” Opt. Lett. 41(15), 3631–3634 (2016).
[Crossref] [PubMed]

L. Gong, W. Liu, Q. Zhao, Y. Ren, X. Qiu, M. Zhong, and Y. Li, “Controllable light capsules employing modified Bessel-Gauss beams,” Sci. Rep. 6(1), 29001 (2016).
[Crossref] [PubMed]

B. R. Patton, D. Burke, D. Owald, T. J. Gould, J. Bewersdorf, and M. J. Booth, “Three-dimensional STED microscopy of aberrating tissue using dual adaptive optics,” Opt. Express 24(8), 8862–8876 (2016).
[Crossref] [PubMed]

2015 (3)

K. Y. Han and T. Ha, “Dual-color three-dimensional STED microscopy with a single high-repetition-rate laser,” Opt. Lett. 40(11), 2653–2656 (2015).
[Crossref] [PubMed]

Z. Zhang, H. Fan, H.-F. Xu, J. Qu, and W. Huang, “Three-dimensional focus shaping of partially coherent circularly polarized vortex beams using a binary optic,” J. Opt. 17(6), 065611 (2015).
[Crossref]

L. Lanzanò, I. Coto Hernández, M. Castello, E. Gratton, A. Diaspro, and G. Vicidomini, “Encoding and decoding spatio-temporal information for super-resolution microscopy,” Nat. Commun. 6(1), 6701 (2015).
[Crossref] [PubMed]

2014 (1)

X. Weng, X. Gao, H. Guo, and S. Zhuang, “Creation of tunable multiple 3D dark spots with cylindrical vector beam,” Appl. Opt. 53(11), 2470–2476 (2014).
[Crossref] [PubMed]

2013 (2)

R. Wollhofen, J. Katzmann, C. Hrelescu, J. Jacak, and T. A. Klar, “120 nm resolution and 55 nm structure size in STED-lithography,” Opt. Express 21(9), 10831–10840 (2013).
[Crossref] [PubMed]

C. Eggeling, K. I. Willig, and F. J. Barrantes, “STED microscopy of living cells--new frontiers in membrane and neurobiology,” J. Neurochem. 126(2), 203–212 (2013).
[Crossref] [PubMed]

2012 (3)

T. Grotjohann, I. Testa, M. Reuss, T. Brakemann, C. Eggeling, S. W. Hell, and S. Jakobs, “rsEGFP2 enables fast RESOLFT nanoscopy of living cells,” eLife 1, e00248 (2012).
[Crossref] [PubMed]

C. Alpmann, M. Esseling, P. Rose, and C. Denz, “Holographic optical bottle beams,” Appl. Phys. Lett. 100(11), 111101 (2012).
[Crossref]

Y. Xue, C. Kuang, S. Li, Z. Gu, and X. Liu, “Sharper fluorescent super-resolution spot generated by azimuthally polarized beam in STED microscopy,” Opt. Express 20(16), 17653–17666 (2012).
[Crossref] [PubMed]

2011 (4)

Y. Xue, C. Kuang, X. Hao, Z. Gu, and X. Liu, “A method for generating a three-dimensional dark spot using a radially polarized beam,” J. Opt. 13(12), 125704 (2011).
[Crossref]

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[Crossref] [PubMed]

T. J. Gould, J. R. Myers, and J. Bewersdorf, “Total internal reflection STED microscopy,” Opt. Express 19(14), 13351–13357 (2011).
[Crossref] [PubMed]

A. M. Yao and M. J. Padgett, “Orbital angular momentum: origins, behavior and applications,” Adv. Opt. Photonics 3(2), 161–204 (2011).
[Crossref]

2010 (3)

Y. Zhang, “Generation of three-dimensional dark spots with a perfect light shell with a radially polarized Laguerre-Gaussian beam,” Appl. Opt. 49(32), 6217–6223 (2010).
[Crossref] [PubMed]

M. Leutenegger, C. Eggeling, and S. W. Hell, “Analytical description of STED microscopy performance,” Opt. Express 18(25), 26417–26429 (2010).
[Crossref] [PubMed]

S. Deng, L. Liu, Y. Cheng, R. Li, and Z. Xu, “Effects of primary aberrations on the fluorescence depletion patterns of STED microscopy,” Opt. Express 18(2), 1657–1666 (2010).
[Crossref] [PubMed]

2009 (2)

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]

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[Crossref] [PubMed]

2008 (4)

A. Punge, S. O. Rizzoli, R. Jahn, J. D. Wildanger, L. Meyer, A. Schönle, L. Kastrup, and S. W. Hell, “3D reconstruction of high-resolution STED microscope images,” Microsc. Res. Tech. 71(9), 644–650 (2008).
[Crossref] [PubMed]

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

B. 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]

Y. Kozawa and S. Sato, “Dark-spot formation by vector beams,” Opt. Lett. 33(20), 2326–2328 (2008).
[Crossref] [PubMed]

2007 (1)

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

2006 (3)

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

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[Crossref] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
[Crossref] [PubMed]

2005 (4)

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. 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]

V. Westphal and S. W. Hell, “Nanoscale resolution in the focal plane of an optical microscope,” Phys. Rev. Lett. 94(14), 143903 (2005).
[Crossref] [PubMed]

L. Kastrup, H. Blom, C. Eggeling, and S. W. Hell, “Fluorescence fluctuation spectroscopy in subdiffraction focal volumes,” Phys. Rev. Lett. 94(17), 178104 (2005).
[Crossref] [PubMed]

2004 (1)

P. Török and P. Munro, “The use of Gauss-Laguerre vector beams in STED microscopy,” Opt. Express 12(15), 3605–3617 (2004).
[Crossref] [PubMed]

2002 (2)

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]

R. Heintzmann, T. M. Jovin, and C. Cremer, “Saturated patterned excitation microscopy--a concept for optical resolution improvement,” J. Opt. Soc. Am. A 19(8), 1599–1609 (2002).
[Crossref] [PubMed]

2000 (2)

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

J. Arlt and M. J. Padgett, “Generation of a beam with a dark focus surrounded by regions of higher intensity: the optical bottle beam,” Opt. Lett. 25(4), 191–193 (2000).
[Crossref] [PubMed]

1994 (1)

S. W. Hell and J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 780–782 (1994).
[Crossref] [PubMed]

1988 (1)

C. Sheppard and Z. Hegedus, “Axial behavior of pupil-plane filters,” J. Opt. Soc. Am. A 5(5), 643–647 (1988).
[Crossref]

1985 (1)

W. Condell, “Fraunhofer diffraction from a circular annular aperture with helical phase factor,” J. Opt. Soc. Am. A 2(2), 206–208 (1985).
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C. Alpmann, M. Esseling, P. Rose, and C. Denz, “Holographic optical bottle beams,” Appl. Phys. Lett. 100(11), 111101 (2012).
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J. Antonello, D. Burke, and M. J. Booth, “Aberrations in stimulated emission depletion (STED) microscopy,” Opt. Commun. 404, 203–209 (2017).
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J. Antonello, E. B. Kromann, D. Burke, J. Bewersdorf, and M. J. Booth, “Coma aberrations in combined two- and three-dimensional STED nanoscopy,” Opt. Lett. 41(15), 3631–3634 (2016).
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J. Arlt and M. J. Padgett, “Generation of a beam with a dark focus surrounded by regions of higher intensity: the optical bottle beam,” Opt. Lett. 25(4), 191–193 (2000).
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F. Balzarotti, Y. Eilers, K. C. Gwosch, A. H. Gynnå, V. Westphal, F. D. Stefani, J. Elf, and S. W. Hell, “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes,” Science 355(6325), 606–612 (2017).
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Barrantes, F. J.

C. Eggeling, K. I. Willig, and F. J. Barrantes, “STED microscopy of living cells--new frontiers in membrane and neurobiology,” J. Neurochem. 126(2), 203–212 (2013).
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M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
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C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
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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).
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Bewersdorf, J.

B. R. Patton, D. Burke, D. Owald, T. J. Gould, J. Bewersdorf, and M. J. Booth, “Three-dimensional STED microscopy of aberrating tissue using dual adaptive optics,” Opt. Express 24(8), 8862–8876 (2016).
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J. Antonello, E. B. Kromann, D. Burke, J. Bewersdorf, and M. J. Booth, “Coma aberrations in combined two- and three-dimensional STED nanoscopy,” Opt. Lett. 41(15), 3631–3634 (2016).
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T. J. Gould, J. R. Myers, and J. Bewersdorf, “Total internal reflection STED microscopy,” Opt. Express 19(14), 13351–13357 (2011).
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L. Kastrup, H. Blom, C. Eggeling, and S. W. Hell, “Fluorescence fluctuation spectroscopy in subdiffraction focal volumes,” Phys. Rev. Lett. 94(17), 178104 (2005).
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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).
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Booth, M. J.

J. Antonello, D. Burke, and M. J. Booth, “Aberrations in stimulated emission depletion (STED) microscopy,” Opt. Commun. 404, 203–209 (2017).
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J. Antonello, E. B. Kromann, D. Burke, J. Bewersdorf, and M. J. Booth, “Coma aberrations in combined two- and three-dimensional STED nanoscopy,” Opt. Lett. 41(15), 3631–3634 (2016).
[Crossref] [PubMed]

B. R. Patton, D. Burke, D. Owald, T. J. Gould, J. Bewersdorf, and M. J. Booth, “Three-dimensional STED microscopy of aberrating tissue using dual adaptive optics,” Opt. Express 24(8), 8862–8876 (2016).
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T. Grotjohann, I. Testa, M. Reuss, T. Brakemann, C. Eggeling, S. W. Hell, and S. Jakobs, “rsEGFP2 enables fast RESOLFT nanoscopy of living cells,” eLife 1, e00248 (2012).
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Burke, D.

J. Antonello, D. Burke, and M. J. Booth, “Aberrations in stimulated emission depletion (STED) microscopy,” Opt. Commun. 404, 203–209 (2017).
[Crossref] [PubMed]

J. Antonello, E. B. Kromann, D. Burke, J. Bewersdorf, and M. J. Booth, “Coma aberrations in combined two- and three-dimensional STED nanoscopy,” Opt. Lett. 41(15), 3631–3634 (2016).
[Crossref] [PubMed]

B. R. Patton, D. Burke, D. Owald, T. J. Gould, J. Bewersdorf, and M. J. Booth, “Three-dimensional STED microscopy of aberrating tissue using dual adaptive optics,” Opt. Express 24(8), 8862–8876 (2016).
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Cao, Y.

B. Wang, J. Shi, T. Zhang, X. Xu, Y. Cao, and X. Li, “Improved lateral resolution with an annular vortex depletion beam in STED microscopy,” Opt. Lett. 42(23), 4885–4888 (2017).
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L. Lanzanò, I. Coto Hernández, M. Castello, E. Gratton, A. Diaspro, and G. Vicidomini, “Encoding and decoding spatio-temporal information for super-resolution microscopy,” Nat. Commun. 6(1), 6701 (2015).
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L. Wang, B. Chen, W. Yan, Z. Yang, X. Peng, D. Lin, X. Weng, T. Ye, and J. Qu, “Resolution improvement in STED super-resolution microscopy at low power using a phasor plot approach,” Nanoscale 10(34), 16252–16260 (2018).
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S. Deng, L. Liu, Y. Cheng, R. Li, and Z. Xu, “Effects of primary aberrations on the fluorescence depletion patterns of STED microscopy,” Opt. Express 18(2), 1657–1666 (2010).
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Condell, W.

W. Condell, “Fraunhofer diffraction from a circular annular aperture with helical phase factor,” J. Opt. Soc. Am. A 2(2), 206–208 (1985).
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Coto Hernández, I.

L. Lanzanò, I. Coto Hernández, M. Castello, E. Gratton, A. Diaspro, and G. Vicidomini, “Encoding and decoding spatio-temporal information for super-resolution microscopy,” Nat. Commun. 6(1), 6701 (2015).
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R. Heintzmann, T. M. Jovin, and C. Cremer, “Saturated patterned excitation microscopy--a concept for optical resolution improvement,” J. Opt. Soc. Am. A 19(8), 1599–1609 (2002).
[Crossref] [PubMed]

D’Este, E.

J. Heine, M. Reuss, B. Harke, E. D’Este, S. J. Sahl, and S. W. Hell, “Adaptive-illumination STED nanoscopy,” Proc. Natl. Acad. Sci. U.S.A. 114(37), 9797–9802 (2017).
[Crossref] [PubMed]

Davidson, M. W.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006).
[Crossref] [PubMed]

Deng, S.

S. Deng, L. Liu, Y. Cheng, R. Li, and Z. Xu, “Effects of primary aberrations on the fluorescence depletion patterns of STED microscopy,” Opt. Express 18(2), 1657–1666 (2010).
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C. Alpmann, M. Esseling, P. Rose, and C. Denz, “Holographic optical bottle beams,” Appl. Phys. Lett. 100(11), 111101 (2012).
[Crossref]

Diaspro, A.

L. Lanzanò, I. Coto Hernández, M. Castello, E. Gratton, A. Diaspro, and G. Vicidomini, “Encoding and decoding spatio-temporal information for super-resolution microscopy,” Nat. Commun. 6(1), 6701 (2015).
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Donnert, G.

J. Heine, C. A. Wurm, J. Keller-Findeisen, A. Schönle, B. Harke, M. Reuss, F. R. Winter, and G. Donnert, “Three dimensional live-cell STED microscopy at increased depth using a water immersion objective,” Rev. Sci. Instrum. 89(5), 053701 (2018).
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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).
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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).
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Eggeling, C.

C. Eggeling, K. I. Willig, and F. J. Barrantes, “STED microscopy of living cells--new frontiers in membrane and neurobiology,” J. Neurochem. 126(2), 203–212 (2013).
[Crossref] [PubMed]

T. Grotjohann, I. Testa, M. Reuss, T. Brakemann, C. Eggeling, S. W. Hell, and S. Jakobs, “rsEGFP2 enables fast RESOLFT nanoscopy of living cells,” eLife 1, e00248 (2012).
[Crossref] [PubMed]

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[Crossref] [PubMed]

M. Leutenegger, C. Eggeling, and S. W. Hell, “Analytical description of STED microscopy performance,” Opt. Express 18(25), 26417–26429 (2010).
[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]

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[Crossref] [PubMed]

L. Kastrup, H. Blom, C. Eggeling, and S. W. Hell, “Fluorescence fluctuation spectroscopy in subdiffraction focal volumes,” Phys. Rev. Lett. 94(17), 178104 (2005).
[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]

Eilers, Y.

F. Balzarotti, Y. Eilers, K. C. Gwosch, A. H. Gynnå, V. Westphal, F. D. Stefani, J. Elf, and S. W. Hell, “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes,” Science 355(6325), 606–612 (2017).
[Crossref] [PubMed]

Elf, J.

F. Balzarotti, Y. Eilers, K. C. Gwosch, A. H. Gynnå, V. Westphal, F. D. Stefani, J. Elf, and S. W. Hell, “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes,” Science 355(6325), 606–612 (2017).
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Engelhardt, J.

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
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Esseling, M.

C. Alpmann, M. Esseling, P. Rose, and C. Denz, “Holographic optical bottle beams,” Appl. Phys. Lett. 100(11), 111101 (2012).
[Crossref]

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Z. Zhang, H. Fan, H.-F. Xu, J. Qu, and W. Huang, “Three-dimensional focus shaping of partially coherent circularly polarized vortex beams using a binary optic,” J. Opt. 17(6), 065611 (2015).
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P. Gao, B. Prunsche, L. Zhou, K. Nienhaus, and G. U. Nienhaus, “Background suppression in fluorescence nanoscopy with stimulated emission double depletion,” Nat. Photonics 11(3), 163–169 (2017).
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Gao, X.

X. Weng, X. Gao, H. Guo, and S. Zhuang, “Creation of tunable multiple 3D dark spots with cylindrical vector beam,” Appl. Opt. 53(11), 2470–2476 (2014).
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S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
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L. Gong, W. Liu, Q. Zhao, Y. Ren, X. Qiu, M. Zhong, and Y. Li, “Controllable light capsules employing modified Bessel-Gauss beams,” Sci. Rep. 6(1), 29001 (2016).
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Görlich, D.

F. Göttfert, T. Pleiner, J. Heine, V. Westphal, D. Görlich, S. J. Sahl, and S. W. Hell, “Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent,” Proc. Natl. Acad. Sci. U.S.A. 114(9), 2125–2130 (2017).
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Göttfert, F.

F. Göttfert, T. Pleiner, J. Heine, V. Westphal, D. Görlich, S. J. Sahl, and S. W. Hell, “Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent,” Proc. Natl. Acad. Sci. U.S.A. 114(9), 2125–2130 (2017).
[Crossref] [PubMed]

Gould, T. J.

B. R. Patton, D. Burke, D. Owald, T. J. Gould, J. Bewersdorf, and M. J. Booth, “Three-dimensional STED microscopy of aberrating tissue using dual adaptive optics,” Opt. Express 24(8), 8862–8876 (2016).
[Crossref] [PubMed]

T. J. Gould, J. R. Myers, and J. Bewersdorf, “Total internal reflection STED microscopy,” Opt. Express 19(14), 13351–13357 (2011).
[Crossref] [PubMed]

Gratton, E.

L. Lanzanò, I. Coto Hernández, M. Castello, E. Gratton, A. Diaspro, and G. Vicidomini, “Encoding and decoding spatio-temporal information for super-resolution microscopy,” Nat. Commun. 6(1), 6701 (2015).
[Crossref] [PubMed]

Grotjohann, T.

T. Grotjohann, I. Testa, M. Reuss, T. Brakemann, C. Eggeling, S. W. Hell, and S. Jakobs, “rsEGFP2 enables fast RESOLFT nanoscopy of living cells,” eLife 1, e00248 (2012).
[Crossref] [PubMed]

Gu, Z.

Y. Xue, C. Kuang, S. Li, Z. Gu, and X. Liu, “Sharper fluorescent super-resolution spot generated by azimuthally polarized beam in STED microscopy,” Opt. Express 20(16), 17653–17666 (2012).
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Y. Xue, C. Kuang, X. Hao, Z. Gu, and X. Liu, “A method for generating a three-dimensional dark spot using a radially polarized beam,” J. Opt. 13(12), 125704 (2011).
[Crossref]

Guo, H.

X. Weng, X. Gao, H. Guo, and S. Zhuang, “Creation of tunable multiple 3D dark spots with cylindrical vector beam,” Appl. Opt. 53(11), 2470–2476 (2014).
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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).
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Gwosch, K. C.

F. Balzarotti, Y. Eilers, K. C. Gwosch, A. H. Gynnå, V. Westphal, F. D. Stefani, J. Elf, and S. W. Hell, “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes,” Science 355(6325), 606–612 (2017).
[Crossref] [PubMed]

Gynnå, A. H.

F. Balzarotti, Y. Eilers, K. C. Gwosch, A. H. Gynnå, V. Westphal, F. D. Stefani, J. Elf, and S. W. Hell, “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes,” Science 355(6325), 606–612 (2017).
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Ha, T.

K. Y. Han and T. Ha, “Dual-color three-dimensional STED microscopy with a single high-repetition-rate laser,” Opt. Lett. 40(11), 2653–2656 (2015).
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Han, K. Y.

K. Y. Han and T. Ha, “Dual-color three-dimensional STED microscopy with a single high-repetition-rate laser,” Opt. Lett. 40(11), 2653–2656 (2015).
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G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (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]

Hao, X.

Y. Xue, C. Kuang, X. Hao, Z. Gu, and X. Liu, “A method for generating a three-dimensional dark spot using a radially polarized beam,” J. Opt. 13(12), 125704 (2011).
[Crossref]

Harke, B.

J. Heine, C. A. Wurm, J. Keller-Findeisen, A. Schönle, B. Harke, M. Reuss, F. R. Winter, and G. Donnert, “Three dimensional live-cell STED microscopy at increased depth using a water immersion objective,” Rev. Sci. Instrum. 89(5), 053701 (2018).
[Crossref] [PubMed]

J. Heine, M. Reuss, B. Harke, E. D’Este, S. J. Sahl, and S. W. Hell, “Adaptive-illumination STED nanoscopy,” Proc. Natl. Acad. Sci. U.S.A. 114(37), 9797–9802 (2017).
[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]

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]

Hegedus, Z.

C. Sheppard and Z. Hegedus, “Axial behavior of pupil-plane filters,” J. Opt. Soc. Am. A 5(5), 643–647 (1988).
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C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[Crossref] [PubMed]

Heine, J.

J. Heine, C. A. Wurm, J. Keller-Findeisen, A. Schönle, B. Harke, M. Reuss, F. R. Winter, and G. Donnert, “Three dimensional live-cell STED microscopy at increased depth using a water immersion objective,” Rev. Sci. Instrum. 89(5), 053701 (2018).
[Crossref] [PubMed]

F. Göttfert, T. Pleiner, J. Heine, V. Westphal, D. Görlich, S. J. Sahl, and S. W. Hell, “Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent,” Proc. Natl. Acad. Sci. U.S.A. 114(9), 2125–2130 (2017).
[Crossref] [PubMed]

J. Heine, M. Reuss, B. Harke, E. D’Este, S. J. Sahl, and S. W. Hell, “Adaptive-illumination STED nanoscopy,” Proc. Natl. Acad. Sci. U.S.A. 114(37), 9797–9802 (2017).
[Crossref] [PubMed]

Heintzmann, R.

R. Heintzmann, T. M. Jovin, and C. Cremer, “Saturated patterned excitation microscopy--a concept for optical resolution improvement,” J. Opt. Soc. Am. A 19(8), 1599–1609 (2002).
[Crossref] [PubMed]

Hell, S. W.

J. Heine, M. Reuss, B. Harke, E. D’Este, S. J. Sahl, and S. W. Hell, “Adaptive-illumination STED nanoscopy,” Proc. Natl. Acad. Sci. U.S.A. 114(37), 9797–9802 (2017).
[Crossref] [PubMed]

F. Göttfert, T. Pleiner, J. Heine, V. Westphal, D. Görlich, S. J. Sahl, and S. W. Hell, “Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent,” Proc. Natl. Acad. Sci. U.S.A. 114(9), 2125–2130 (2017).
[Crossref] [PubMed]

F. Balzarotti, Y. Eilers, K. C. Gwosch, A. H. Gynnå, V. Westphal, F. D. Stefani, J. Elf, and S. W. Hell, “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes,” Science 355(6325), 606–612 (2017).
[Crossref] [PubMed]

T. Grotjohann, I. Testa, M. Reuss, T. Brakemann, C. Eggeling, S. W. Hell, and S. Jakobs, “rsEGFP2 enables fast RESOLFT nanoscopy of living cells,” eLife 1, e00248 (2012).
[Crossref] [PubMed]

G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
[Crossref] [PubMed]

M. Leutenegger, C. Eggeling, and S. W. Hell, “Analytical description of STED microscopy performance,” Opt. Express 18(25), 26417–26429 (2010).
[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]

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
[Crossref] [PubMed]

A. Punge, S. O. Rizzoli, R. Jahn, J. D. Wildanger, L. Meyer, A. Schönle, L. Kastrup, and S. W. Hell, “3D reconstruction of high-resolution STED microscope images,” Microsc. Res. Tech. 71(9), 644–650 (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]

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]

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

M. 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]

V. Westphal and S. W. Hell, “Nanoscale resolution in the focal plane of an optical microscope,” Phys. Rev. Lett. 94(14), 143903 (2005).
[Crossref] [PubMed]

L. Kastrup, H. Blom, C. Eggeling, and S. W. Hell, “Fluorescence fluctuation spectroscopy in subdiffraction focal volumes,” Phys. Rev. Lett. 94(17), 178104 (2005).
[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 J. Wichmann, “Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy,” Opt. Lett. 19(11), 780–782 (1994).
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L. Lanzanò, I. Coto Hernández, M. Castello, E. Gratton, A. Diaspro, and G. Vicidomini, “Encoding and decoding spatio-temporal information for super-resolution microscopy,” Nat. Commun. 6(1), 6701 (2015).
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G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
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von Middendorff, C.

C. Eggeling, C. Ringemann, R. Medda, G. Schwarzmann, K. Sandhoff, S. Polyakova, V. N. Belov, B. Hein, C. von Middendorff, A. Schönle, and S. W. Hell, “Direct observation of the nanoscale dynamics of membrane lipids in a living cell,” Nature 457(7233), 1159–1162 (2009).
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Wang, B.

B. Wang, J. Shi, T. Zhang, X. Xu, Y. Cao, and X. Li, “Improved lateral resolution with an annular vortex depletion beam in STED microscopy,” Opt. Lett. 42(23), 4885–4888 (2017).
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Wang, L.

L. Wang, B. Chen, W. Yan, Z. Yang, X. Peng, D. Lin, X. Weng, T. Ye, and J. Qu, “Resolution improvement in STED super-resolution microscopy at low power using a phasor plot approach,” Nanoscale 10(34), 16252–16260 (2018).
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Warburton, R. J.

T. Kaldewey, A. V. Kuhlmann, S. R. Valentin, A. Ludwig, A. D. Wieck, and R. J. Warburton, “Far-field nanoscopy on a semiconductor quantum dot via a rapid-adiabatic-passage-based switch,” Nat. Photonics 12(2), 68–72 (2018).
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Weng, X.

L. Wang, B. Chen, W. Yan, Z. Yang, X. Peng, D. Lin, X. Weng, T. Ye, and J. Qu, “Resolution improvement in STED super-resolution microscopy at low power using a phasor plot approach,” Nanoscale 10(34), 16252–16260 (2018).
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X. Weng, X. Gao, H. Guo, and S. Zhuang, “Creation of tunable multiple 3D dark spots with cylindrical vector beam,” Appl. Opt. 53(11), 2470–2476 (2014).
[Crossref] [PubMed]

Westphal, V.

F. Göttfert, T. Pleiner, J. Heine, V. Westphal, D. Görlich, S. J. Sahl, and S. W. Hell, “Strong signal increase in STED fluorescence microscopy by imaging regions of subdiffraction extent,” Proc. Natl. Acad. Sci. U.S.A. 114(9), 2125–2130 (2017).
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F. Balzarotti, Y. Eilers, K. C. Gwosch, A. H. Gynnå, V. Westphal, F. D. Stefani, J. Elf, and S. W. Hell, “Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes,” Science 355(6325), 606–612 (2017).
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G. Vicidomini, G. Moneron, K. Y. Han, V. Westphal, H. Ta, M. Reuss, J. Engelhardt, C. Eggeling, and S. W. Hell, “Sharper low-power STED nanoscopy by time gating,” Nat. Methods 8(7), 571–573 (2011).
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T. Kaldewey, A. V. Kuhlmann, S. R. Valentin, A. Ludwig, A. D. Wieck, and R. J. Warburton, “Far-field nanoscopy on a semiconductor quantum dot via a rapid-adiabatic-passage-based switch,” Nat. Photonics 12(2), 68–72 (2018).
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A. Punge, S. O. Rizzoli, R. Jahn, J. D. Wildanger, L. Meyer, A. Schönle, L. Kastrup, and S. W. Hell, “3D reconstruction of high-resolution STED microscope images,” Microsc. Res. Tech. 71(9), 644–650 (2008).
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C. Eggeling, K. I. Willig, and F. J. Barrantes, “STED microscopy of living cells--new frontiers in membrane and neurobiology,” J. Neurochem. 126(2), 203–212 (2013).
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J. Heine, C. A. Wurm, J. Keller-Findeisen, A. Schönle, B. Harke, M. Reuss, F. R. Winter, and G. Donnert, “Three dimensional live-cell STED microscopy at increased depth using a water immersion objective,” Rev. Sci. Instrum. 89(5), 053701 (2018).
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B. Richards and E. Wolf, “Electromagnetic Diffraction in Optical Systems. 2. Structure of the Image Field in an Aplanatic System,” Proc R Soc Lon Ser-A253, 358–379 (1959).

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R. Wollhofen, J. Katzmann, C. Hrelescu, J. Jacak, and T. A. Klar, “120 nm resolution and 55 nm structure size in STED-lithography,” Opt. Express 21(9), 10831–10840 (2013).
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J. Heine, C. A. Wurm, J. Keller-Findeisen, A. Schönle, B. Harke, M. Reuss, F. R. Winter, and G. Donnert, “Three dimensional live-cell STED microscopy at increased depth using a water immersion objective,” Rev. Sci. Instrum. 89(5), 053701 (2018).
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Z. Zhang, H. Fan, H.-F. Xu, J. Qu, and W. Huang, “Three-dimensional focus shaping of partially coherent circularly polarized vortex beams using a binary optic,” J. Opt. 17(6), 065611 (2015).
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B. Wang, J. Shi, T. Zhang, X. Xu, Y. Cao, and X. Li, “Improved lateral resolution with an annular vortex depletion beam in STED microscopy,” Opt. Lett. 42(23), 4885–4888 (2017).
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Xu, Z.

S. Deng, L. Liu, Y. Cheng, R. Li, and Z. Xu, “Effects of primary aberrations on the fluorescence depletion patterns of STED microscopy,” Opt. Express 18(2), 1657–1666 (2010).
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Y. Xue, C. Kuang, S. Li, Z. Gu, and X. Liu, “Sharper fluorescent super-resolution spot generated by azimuthally polarized beam in STED microscopy,” Opt. Express 20(16), 17653–17666 (2012).
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Yang, Z.

L. Wang, B. Chen, W. Yan, Z. Yang, X. Peng, D. Lin, X. Weng, T. Ye, and J. Qu, “Resolution improvement in STED super-resolution microscopy at low power using a phasor plot approach,” Nanoscale 10(34), 16252–16260 (2018).
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B. Wang, J. Shi, T. Zhang, X. Xu, Y. Cao, and X. Li, “Improved lateral resolution with an annular vortex depletion beam in STED microscopy,” Opt. Lett. 42(23), 4885–4888 (2017).
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L. Gong, W. Liu, Q. Zhao, Y. Ren, X. Qiu, M. Zhong, and Y. Li, “Controllable light capsules employing modified Bessel-Gauss beams,” Sci. Rep. 6(1), 29001 (2016).
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Adv. Opt. Photonics (1)

A. M. Yao and M. J. Padgett, “Orbital angular momentum: origins, behavior and applications,” Adv. Opt. Photonics 3(2), 161–204 (2011).
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Appl. Opt. (2)

Y. Zhang, “Generation of three-dimensional dark spots with a perfect light shell with a radially polarized Laguerre-Gaussian beam,” Appl. Opt. 49(32), 6217–6223 (2010).
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X. Weng, X. Gao, H. Guo, and S. Zhuang, “Creation of tunable multiple 3D dark spots with cylindrical vector beam,” Appl. Opt. 53(11), 2470–2476 (2014).
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J. Neurochem. (1)

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Nano Lett. (1)

B. Harke, C. K. Ullal, J. Keller, and S. W. Hell, “Three-dimensional nanoscopy of colloidal crystals,” Nano Lett. 8(5), 1309–1313 (2008).
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Nanoscale (1)

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Nat. Commun. (1)

L. Lanzanò, I. Coto Hernández, M. Castello, E. Gratton, A. Diaspro, and G. Vicidomini, “Encoding and decoding spatio-temporal information for super-resolution microscopy,” Nat. Commun. 6(1), 6701 (2015).
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Nat. Methods (2)

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T. Kaldewey, A. V. Kuhlmann, S. R. Valentin, A. Ludwig, A. D. Wieck, and R. J. Warburton, “Far-field nanoscopy on a semiconductor quantum dot via a rapid-adiabatic-passage-based switch,” Nat. Photonics 12(2), 68–72 (2018).
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Nature (1)

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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).
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J. Keller, A. Schönle, and S. W. Hell, “Efficient fluorescence inhibition patterns for RESOLFT microscopy,” Opt. Express 15(6), 3361–3371 (2007).
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P. Török and P. Munro, “The use of Gauss-Laguerre vector beams in STED microscopy,” Opt. Express 12(15), 3605–3617 (2004).
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S. Deng, L. Liu, Y. Cheng, R. Li, and Z. Xu, “Effects of primary aberrations on the fluorescence depletion patterns of STED microscopy,” Opt. Express 18(2), 1657–1666 (2010).
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B. R. Patton, D. Burke, D. Owald, T. J. Gould, J. Bewersdorf, and M. J. Booth, “Three-dimensional STED microscopy of aberrating tissue using dual adaptive optics,” Opt. Express 24(8), 8862–8876 (2016).
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M. Leutenegger, C. Eggeling, and S. W. Hell, “Analytical description of STED microscopy performance,” Opt. Express 18(25), 26417–26429 (2010).
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Y. Xue, C. Kuang, S. Li, Z. Gu, and X. Liu, “Sharper fluorescent super-resolution spot generated by azimuthally polarized beam in STED microscopy,” Opt. Express 20(16), 17653–17666 (2012).
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K. Y. Han and T. Ha, “Dual-color three-dimensional STED microscopy with a single high-repetition-rate laser,” Opt. Lett. 40(11), 2653–2656 (2015).
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J. Antonello, E. B. Kromann, D. Burke, J. Bewersdorf, and M. J. Booth, “Coma aberrations in combined two- and three-dimensional STED nanoscopy,” Opt. Lett. 41(15), 3631–3634 (2016).
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B. Wang, J. Shi, T. Zhang, X. Xu, Y. Cao, and X. Li, “Improved lateral resolution with an annular vortex depletion beam in STED microscopy,” Opt. Lett. 42(23), 4885–4888 (2017).
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Optica (1)

K. Sozanski, E. Sisamakis, X. Zhang, and R. Holyst, “Quantitative fluorescence correlation spectroscopy in three-dimensional systems under stimulated emission depletion conditions,” Optica 4(8), 982–988 (2017).
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V. Westphal and S. W. Hell, “Nanoscale resolution in the focal plane of an optical microscope,” Phys. Rev. Lett. 94(14), 143903 (2005).
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J. Heine, C. A. Wurm, J. Keller-Findeisen, A. Schönle, B. Harke, M. Reuss, F. R. Winter, and G. Donnert, “Three dimensional live-cell STED microscopy at increased depth using a water immersion objective,” Rev. Sci. Instrum. 89(5), 053701 (2018).
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Sci. Rep. (1)

L. Gong, W. Liu, Q. Zhao, Y. Ren, X. Qiu, M. Zhong, and Y. Li, “Controllable light capsules employing modified Bessel-Gauss beams,” Sci. Rep. 6(1), 29001 (2016).
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Supplementary Material (2)

NameDescription
» Visualization 1       Raw data (z-stack acquisition) used to generate the chromo-projection in Fig. 4c.
» Visualization 2       Large field-of-view raw data (z-stack acquisition) of tubulin-labeled Indian muntjac cells. Left: 2D-STED (30mW), Center: 2D-STED (80mW); Right: CH-STED (80mW, rho=0.86). Slice distance is 200nm.

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Figures (9)

Fig. 1
Fig. 1 Phase masks for the standard STED modes and for a ‘radial vortex’. a) z-STED mask for mostly-axial confinement. b) 2D-STED mask for transverse confinement. c) An intensity-zero is warranted whenever a vortex-phase is added to an arbitrary radial-only function, f(r) (Appendix A). Off-axis radial phase gradients (Δ=0) can be exploited to generate an axial gradient for STED confinement. The bottom rows show experimental cross-sections and focal profiles of z-STED and 2D-STED using gold-bead scattering (775nm wavelength, 1.4 NA objective) with corresponding x-y imaging of microtubule filaments.
Fig. 2
Fig. 2 (a) Simplified layout of the modified STED setup. (b) Interpretation and observation by gold bead scattering of the tunable dip generation. The scattering images are for an xz-scanned gold bead displayed with a linear and a saturated look-up table (LUT), the latter providing a heuristic preview of the effective fluorescence source at high saturation. (c) Data-points and paraxial theory (solid lines) for the depletion beam focal plane profile. In the inset, beam’s geometrical confinement metric (second-order derivative of intensity) with experimental data and theory as a function of the bi-vortex radius, ρ. The single adjusted parameter (both in the main graph and in the inset) is a global vertical normalization factor.
Fig. 3
Fig. 3 STED modes PSFs. (a) Nano-bead fluorescence xz-scans in different STED modes (same LUT display). The inset highlights the origin of the excited ghosts that are poorly depleted by the z-STED beam. (b) Left: gold bead scattering cross-section in 2D-STED and CH-STED with isophote lines defining saturation contours. White-to-red represent signal rescue in 2D-STED (depletion power change) and in CH-STED (ρ change). Red and white isophotes in the CH-STED panel are for the same intensity value and are therefore representative of an actual 2D- to CH-STED transition. Right: effective PSF in the two STED regimes. Red-blue images (same LUT) are log-intensity versions that highlight background noise.
Fig. 4
Fig. 4 2D-STED to CH-STED transition in constant-power mode with all acquisition parameters (apart from the phase mask) kept constant. (a) Single-slice and projections from a z-stack acquisition of a tubulin-labeled Indian muntjac dividing cell in metaphase, with a kinetochore marker shown in the right panel (red). Top- and bottom-halves are independent, vertically adjacent, acquisitions. (b) Low-signal Indian muntjac dividing cell in anaphase (chromosome staining in the inset) and zoomed ROI at the 2D to CH transition zone. Photon standard deviation, average and their ratio for the top and bottom halves are displayed. (c) Chromo-projection from a z-stack (Visualization 1) of a tubulin-labeled U2OS interphase cell. Color definition is a readout for optical sectioning. (d) Indian muntjac mitotic spindle in all single-beam and incoherent superposition modes. In (c) and (d), where a single object is consecutively imaged, a left-right acquisition sequence is followed.
Fig. 5
Fig. 5 CH-STED vs. 2D-STED axial confinement and background suppression using 40-nm fluorescent beads. (a) PSF lateral dimension using the full-width half-maximum (FWHM) criteria (mean ± s.d., n = 10 beads per datapoint) at and away from the focal plane in 2D-STED as a function of STED laser average power (at back focal plane). In the inset, metrics for assessing performance, where the defocused plane chosen for measuring confinement is at a Rayleigh range distance (zR = 260nm) from the focal plane. (b) PSF lateral dimension (mean ± s.d., n = 10 beads per datapoint) at and away from the focal plane in CH-STED as a function of ρ, using an intermediate-range STED power (60mW). (c) Scatter plot comparison of 2D-STED and CH-STED (at 60mW and 200mW) using a common parameter (D0) as the independent variable. Each datapoint represents one bead. Axes were cropped at 400nm, leaving three 2D-STED (red) datapoints not displayed (used and accounted for in quantifications in (a). (d) Relative background suppression estimate using the focal plane Gaussian curve fit amplitude relative to an average background value at a one wavelength distance from the focal plane (as displayed in (a) inset).
Fig. 6
Fig. 6 (a) Left: STED beam cross-sections with contour lines showing signal rescue (path A) followed by resolution rescue (path B); Right: Surface displaying theoretical resolution under the parabolic approximation (Eq. (2)). Example trajectories for the three base modes are outlined: constant-power (A or A’), constant-geometry (B or the 2D-STED case B’) and constant-resolution (green line). (b) Projection of an anaphase spindle in a tubulin filament-labeled U2OS cell. Blue-red insets are pictorial examples of the PSF cross-section in each condition (taken from Fig. 3(b)) (c) Single slice image of astral microtubules in a prometaphase U2OS cell showing suppression of defocused portions in CH-STED. (d) Central region of a tubulin-labeled Indian muntjac mitotic spindle. Photocount profiles (right) correspond to the yellow dashed lines. (e) Tubulin-labeled Indian muntjac mitotic spindle along alternative paths in the theoretical surface. Independent LUTs are used. All panels in this figure follow a left-right acquisition sequence, with all unstated settings kept constant.
Fig. 7
Fig. 7 Intensity variation of the bi-vortex focal plane profile near the point at which the on-axis curvature vanishes, together with the leading term in the Taylor expansion (parabolic approximation).
Fig. 8
Fig. 8 Predicted variation of the focal plane fluorescence spot size as the CH-STED parameter ρ is varied. The parabolic approximation deviates significantly from the experimental values for values of ρ below 0.9.
Fig. 9
Fig. 9 (a) Actin-labeled axon. Transition to CH-STED displays mostly a loss in lateral resolution. In these thin structures (<λ) SBR improvement is less significant. (b) Standard and CH-STED modes compared to confocal in a cross-section acquisition of a spectrin-labeled neuron cell body. (c) Transition between STED modes during scanning (vertical slow axis) following the paths outlined in Fig. 6(a). The object is the peripheral area of an actin-labeled neuron cell body. (d) Confocal and dual-channel STED imaging of nuclear pore components in HeLa cells.

Equations (14)

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lim x0 I bivortex I 0 ( 2πNA 3λ x ) 2 ( 2 ρ 3 1 ) 2 ,
R λ 2NA 1+ ( 12 ρ 3 ) 2 P STED P SAT ,
[ E + ( s,φ,z ) E ( s,φ,z ) E z ( s,φ,z ) ]= iA 2π 0 α dθ 0 2π dϕ cos 1/2 θsinθM( θ,ϕ ) ×[ ( cosθ1 )exp( 2iϕ ) cosθ+1 2 sinθexp( iϕ ) ] ×exp{ ik[ zcosθ+ssinθcos( ϕφ ) ] }
0 2π dϕ exp(ig(θ)+inϕ)[ ( cosθ1 )exp( 2iϕ ) cosθ+1 2 sinθexp( iϕ ) ]. ×exp{ ik[ zcosθ+ssinθcos( ϕφ ) ] }
exp(iqsinx)= m= J m ( q )exp( imx ),
2πexp[ ig(θ)+ikzcosθ ] ×[ ( cosθ1 ) e i( n+2 )φ J n+2 ( kssinθ ) ( cosθ+1 ) e inφ J n ( kssinθ ) 2 sinθ e i( n+1 )φ J n+1 ( kssinθ ) ]
U f ( u )= exp[ i(k/2f) | u | 2 ] iλf ×A d 2 x M( x )exp[ i k f xu ]
M bV ( x )={ e iθ 0rρR e iθ ρR<rR ,0r>R
M sV ( R;s,ϕ )= d 2 x M sV ( x )exp[ i k f xu ], =i e iφ π 2 fR ks ×[ J 1 ( ksR f ) H 0 ( ksR f ) J 0 ( ksR f ) H 1 ( ksR f ) ]
M bV ( ρ,R;s,ϕ )= M sV ( R;s,ϕ )2 M sV ( ρR;s,ϕ ).
lim s0 I bivortex ( ρ,R;s ) I 0 ( 2πNA 3 s ) 2 ( 2 ρ 3 1 ) 2 .
( FWHM ) 2 = ( FWH M 0 ) 2 1+P/ P Sat + B 2 .
I CHSTED ( λ ex, λ STED ,ρ,R;s )= I ex ( λ ex ,R;s ) 1+α I bV ( λ STED ,ρ,R;s )
I bV ( λ STED ,ρ,R;s )= I 0 (2π R 2 ) 2 | M bV ( λ STED ,ρ,R;s ) | 2 .

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