Abstract

We developed two-photon excitation stimulated emission depletion (STED) nanoscopy using high-peak-power sub-nanosecond 655-nm pulses. The STED pulse exhibited ideal optical properties and sufficient pulse energy to realize a 70-nm spatial resolution in the compact setup with electrically controllable components. For biological applications, we screened suitable fluorescent dyes or proteins and realized the sub-100 nm spatial resolution imaging of presynaptic protein clusters in fixed primary cultured neurons without severe photobleaching. We expect this method to enable visualization of ultrastructures and the cluster dynamics of biomolecules representing physiological functions in living cells and tissue.

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

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  1. W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
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    [Crossref] [PubMed]
  4. R. Kawakami, K. Sawada, Y. Kusama, Y.-C. Fang, S. Kanazawa, Y. Kozawa, S. Sato, H. Yokoyama, and T. Nemoto, “In vivo two-photon imaging of mouse hippocampal neurons in dentate gyrus using a light source based on a high-peak power gain-switched laser diode,” Biomed. Opt. Express 6(3), 891–901 (2015).
    [Crossref] [PubMed]
  5. S. W. Hell, “Far-field optical nanoscopy,” Science 316(5828), 1153–1158 (2007).
    [Crossref] [PubMed]
  6. L. Schermelleh, R. Heintzmann, and H. Leonhardt, “A guide to super-resolution fluorescence microscopy,” J. Cell Biol. 190(2), 165–175 (2010).
    [Crossref] [PubMed]
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    [Crossref] [PubMed]
  8. Q. Li, S. S. H. Wu, and K. C. Chou, “Subdiffraction-limit two-photon fluorescence microscopy for GFP-tagged cell imaging,” Biophys. J. 97(12), 3224–3228 (2009).
    [Crossref] [PubMed]
  9. P. Bianchini, B. Harke, S. Galiani, G. Vicidomini, and A. Diaspro, “Single-wavelength two-photon excitation-stimulated emission depletion (SW2PE-STED) superresolution imaging,” Proc. Natl. Acad. Sci. U.S.A. 109(17), 6390–6393 (2012).
    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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  12. P. W. Winter, A. G. York, D. D. Nogare, M. Ingaramo, R. Christensen, A. Chitnis, G. H. Patterson, and H. Shroff, “Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples,” Optica 1(3), 181–191 (2014).
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    [Crossref] [PubMed]
  16. J.-H. Hung, K. Sato, Y.-C. Fang, L.-H. Peng, T. Nemoto, and H. Yokoyama, “Generation of high-peak-power sub-nanosecond 650-nm-band optical pulses based on semiconductor-laser-controlling technologies,” Appl. Phys. Express 10(10), 102701 (2017).
    [Crossref]
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    [Crossref] [PubMed]
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    [Crossref]
  19. M. Castello, G. Tortarolo, I. Coto Hernández, T. Deguchi, A. Diaspro, and G. Vicidomini, “Removal of anti-Stokes emission background in STED microscopy by FPGA-based synchronous detection,” Rev. Sci. Instrum. 88(5), 053701 (2017).
    [Crossref] [PubMed]
  20. V. Westphal and S. W. Hell, “Nanoscale Resolution in the Focal Plane of an Optical Microscope,” Phys. Rev. Lett. 94(14), 143903 (2005).
  21. T. Ghelani and S. J. Sigrist, “Coupling the Structural and Functional Assembly of Synaptic Release Sites,” Front. Neuroanat. 12, 81 (2018).
    [Crossref] [PubMed]
  22. S. Schrof, T. Staudt, E. Rittweger, N. Wittenmayer, T. Dresbach, J. Engelhardt, and S. W. Hell, “STED nanoscopy with mass-produced laser diodes,” Opt. Express 19(9), 8066–8072 (2011).
    [Crossref] [PubMed]
  23. R. G. Zhai, H. Vardinon-Friedman, C. Cases-Langhoff, B. Becker, E. D. Gundelfinger, N. E. Ziv, and C. C. Garner, “Assembling the Presynaptic Active Zone: A Characterization of an Active One Precursor Vesicle,” Neuron 29(1), 131–143 (2001).
    [Crossref] [PubMed]
  24. 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]
  25. M. Castello, G. Tortarolo, I. C. Hernández, P. Bianchini, M. Buttafava, G. Boso, A. Tosi, A. Diaspro, and G. Vicidomini, “Gated-sted microscopy with subnanosecond pulsed fiber laser for reducing photobleaching,” Microsc. Res. Tech. 79(9), 785–791 (2016).
    [Crossref] [PubMed]
  26. J. Oracz, V. Westphal, C. Radzewicz, S. J. Sahl, and S. W. Hell, “Photobleaching in STED nanoscopy and its dependence on the photon flux applied for reversible silencing of the fluorophore,” Sci. Rep. 7(1), 11354 (2017).
    [Crossref] [PubMed]

2018 (3)

K. Sawada, R. Kawakami, Y.-C. Fang, J.-H. Hung, Y. Kozawa, K. Otomo, S. Sato, H. Yokoyama, and T. Nemoto, “Improvement of two-photon microscopic imaging in deep regions of living mouse brains by utilizing a light source based on an electrically controllable gain-switched laser diode,” Proc. SPIE 10498, 104982L (2018).
[Crossref]

T. Ghelani and S. J. Sigrist, “Coupling the Structural and Functional Assembly of Synaptic Release Sites,” Front. Neuroanat. 12, 81 (2018).
[Crossref] [PubMed]

K. Otomo, T. Hibi, Y.-C. Fang, J.-H. Hung, M. Tsutsumi, R. Kawakami, H. Yokoyama, and T. Nemoto, “Advanced easySTED microscopy based on two-photon excitation by electrical modulations of light pulse wavefronts,” Biomed. Opt. Express 9(6), 2671–2680 (2018).
[Crossref] [PubMed]

2017 (3)

M. Castello, G. Tortarolo, I. Coto Hernández, T. Deguchi, A. Diaspro, and G. Vicidomini, “Removal of anti-Stokes emission background in STED microscopy by FPGA-based synchronous detection,” Rev. Sci. Instrum. 88(5), 053701 (2017).
[Crossref] [PubMed]

J. Oracz, V. Westphal, C. Radzewicz, S. J. Sahl, and S. W. Hell, “Photobleaching in STED nanoscopy and its dependence on the photon flux applied for reversible silencing of the fluorophore,” Sci. Rep. 7(1), 11354 (2017).
[Crossref] [PubMed]

J.-H. Hung, K. Sato, Y.-C. Fang, L.-H. Peng, T. Nemoto, and H. Yokoyama, “Generation of high-peak-power sub-nanosecond 650-nm-band optical pulses based on semiconductor-laser-controlling technologies,” Appl. Phys. Express 10(10), 102701 (2017).
[Crossref]

2016 (2)

I. Coto Hernández, M. Castello, L. Lanzanò, M. d’Amora, P. Bianchini, A. Diaspro, and G. Vicidomini, “Two-Photon Excitation STED Microscopy with Time-Gated Detection,” Sci. Rep. 6(1), 19419 (2016).
[Crossref] [PubMed]

M. Castello, G. Tortarolo, I. C. Hernández, P. Bianchini, M. Buttafava, G. Boso, A. Tosi, A. Diaspro, and G. Vicidomini, “Gated-sted microscopy with subnanosecond pulsed fiber laser for reducing photobleaching,” Microsc. Res. Tech. 79(9), 785–791 (2016).
[Crossref] [PubMed]

2015 (1)

2014 (3)

2013 (4)

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]

R. Kawakami, K. Sawada, A. Sato, T. Hibi, Y. Kozawa, S. Sato, H. Yokoyama, and T. Nemoto, “Visualizing hippocampal neurons with in vivo two-photon microscopy using a 1030 nm picosecond pulse laser,” Sci. Rep. 3(1), 1014 (2013).
[Crossref] [PubMed]

K. T. Takasaki, J. B. Ding, and B. L. Sabatini, “Live-Cell Superresolution Imaging by Pulsed STED Two-Photon Excitation Microscopy,” Biophys. J. 104(4), 770–777 (2013).
[Crossref] [PubMed]

P. Bethge, R. Chéreau, E. Avignone, G. Marsicano, and U. V. Nägerl, “Two-photon excitation STED microscopy in two colors in acute brain slices,” Biophys. J. 104(4), 778–785 (2013).
[Crossref] [PubMed]

2012 (1)

P. Bianchini, B. Harke, S. Galiani, G. Vicidomini, and A. Diaspro, “Single-wavelength two-photon excitation-stimulated emission depletion (SW2PE-STED) superresolution imaging,” Proc. Natl. Acad. Sci. U.S.A. 109(17), 6390–6393 (2012).
[Crossref] [PubMed]

2011 (1)

2010 (1)

L. Schermelleh, R. Heintzmann, and H. Leonhardt, “A guide to super-resolution fluorescence microscopy,” J. Cell Biol. 190(2), 165–175 (2010).
[Crossref] [PubMed]

2009 (2)

Q. Li, S. S. H. Wu, and K. C. Chou, “Subdiffraction-limit two-photon fluorescence microscopy for GFP-tagged cell imaging,” Biophys. J. 97(12), 3224–3228 (2009).
[Crossref] [PubMed]

G. Moneron and S. W. Hell, “Two-photon excitation STED microscopy,” Opt. Express 17(17), 14567–14573 (2009).
[Crossref] [PubMed]

2007 (1)

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

2006 (1)

K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron 50(6), 823–839 (2006).
[Crossref] [PubMed]

2005 (1)

V. Westphal and S. W. Hell, “Nanoscale Resolution in the Focal Plane of an Optical Microscope,” Phys. Rev. Lett. 94(14), 143903 (2005).

2003 (1)

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[Crossref] [PubMed]

2001 (1)

R. G. Zhai, H. Vardinon-Friedman, C. Cases-Langhoff, B. Becker, E. D. Gundelfinger, N. E. Ziv, and C. C. Garner, “Assembling the Presynaptic Active Zone: A Characterization of an Active One Precursor Vesicle,” Neuron 29(1), 131–143 (2001).
[Crossref] [PubMed]

Avignone, E.

P. Bethge, R. Chéreau, E. Avignone, G. Marsicano, and U. V. Nägerl, “Two-photon excitation STED microscopy in two colors in acute brain slices,” Biophys. J. 104(4), 778–785 (2013).
[Crossref] [PubMed]

Becker, B.

R. G. Zhai, H. Vardinon-Friedman, C. Cases-Langhoff, B. Becker, E. D. Gundelfinger, N. E. Ziv, and C. C. Garner, “Assembling the Presynaptic Active Zone: A Characterization of an Active One Precursor Vesicle,” Neuron 29(1), 131–143 (2001).
[Crossref] [PubMed]

Bethge, P.

P. Bethge, R. Chéreau, E. Avignone, G. Marsicano, and U. V. Nägerl, “Two-photon excitation STED microscopy in two colors in acute brain slices,” Biophys. J. 104(4), 778–785 (2013).
[Crossref] [PubMed]

Bianchini, P.

I. Coto Hernández, M. Castello, L. Lanzanò, M. d’Amora, P. Bianchini, A. Diaspro, and G. Vicidomini, “Two-Photon Excitation STED Microscopy with Time-Gated Detection,” Sci. Rep. 6(1), 19419 (2016).
[Crossref] [PubMed]

M. Castello, G. Tortarolo, I. C. Hernández, P. Bianchini, M. Buttafava, G. Boso, A. Tosi, A. Diaspro, and G. Vicidomini, “Gated-sted microscopy with subnanosecond pulsed fiber laser for reducing photobleaching,” Microsc. Res. Tech. 79(9), 785–791 (2016).
[Crossref] [PubMed]

P. Bianchini, B. Harke, S. Galiani, G. Vicidomini, and A. Diaspro, “Single-wavelength two-photon excitation-stimulated emission depletion (SW2PE-STED) superresolution imaging,” Proc. Natl. Acad. Sci. U.S.A. 109(17), 6390–6393 (2012).
[Crossref] [PubMed]

Boso, G.

M. Castello, G. Tortarolo, I. C. Hernández, P. Bianchini, M. Buttafava, G. Boso, A. Tosi, A. Diaspro, and G. Vicidomini, “Gated-sted microscopy with subnanosecond pulsed fiber laser for reducing photobleaching,” Microsc. Res. Tech. 79(9), 785–791 (2016).
[Crossref] [PubMed]

Buttafava, M.

M. Castello, G. Tortarolo, I. C. Hernández, P. Bianchini, M. Buttafava, G. Boso, A. Tosi, A. Diaspro, and G. Vicidomini, “Gated-sted microscopy with subnanosecond pulsed fiber laser for reducing photobleaching,” Microsc. Res. Tech. 79(9), 785–791 (2016).
[Crossref] [PubMed]

Cases-Langhoff, C.

R. G. Zhai, H. Vardinon-Friedman, C. Cases-Langhoff, B. Becker, E. D. Gundelfinger, N. E. Ziv, and C. C. Garner, “Assembling the Presynaptic Active Zone: A Characterization of an Active One Precursor Vesicle,” Neuron 29(1), 131–143 (2001).
[Crossref] [PubMed]

Castello, M.

M. Castello, G. Tortarolo, I. Coto Hernández, T. Deguchi, A. Diaspro, and G. Vicidomini, “Removal of anti-Stokes emission background in STED microscopy by FPGA-based synchronous detection,” Rev. Sci. Instrum. 88(5), 053701 (2017).
[Crossref] [PubMed]

I. Coto Hernández, M. Castello, L. Lanzanò, M. d’Amora, P. Bianchini, A. Diaspro, and G. Vicidomini, “Two-Photon Excitation STED Microscopy with Time-Gated Detection,” Sci. Rep. 6(1), 19419 (2016).
[Crossref] [PubMed]

M. Castello, G. Tortarolo, I. C. Hernández, P. Bianchini, M. Buttafava, G. Boso, A. Tosi, A. Diaspro, and G. Vicidomini, “Gated-sted microscopy with subnanosecond pulsed fiber laser for reducing photobleaching,” Microsc. Res. Tech. 79(9), 785–791 (2016).
[Crossref] [PubMed]

Chéreau, R.

P. Bethge, R. Chéreau, E. Avignone, G. Marsicano, and U. V. Nägerl, “Two-photon excitation STED microscopy in two colors in acute brain slices,” Biophys. J. 104(4), 778–785 (2013).
[Crossref] [PubMed]

Chitnis, A.

Chou, K. C.

Q. Li, S. S. H. Wu, and K. C. Chou, “Subdiffraction-limit two-photon fluorescence microscopy for GFP-tagged cell imaging,” Biophys. J. 97(12), 3224–3228 (2009).
[Crossref] [PubMed]

Christensen, R.

Coto Hernández, I.

M. Castello, G. Tortarolo, I. Coto Hernández, T. Deguchi, A. Diaspro, and G. Vicidomini, “Removal of anti-Stokes emission background in STED microscopy by FPGA-based synchronous detection,” Rev. Sci. Instrum. 88(5), 053701 (2017).
[Crossref] [PubMed]

I. Coto Hernández, M. Castello, L. Lanzanò, M. d’Amora, P. Bianchini, A. Diaspro, and G. Vicidomini, “Two-Photon Excitation STED Microscopy with Time-Gated Detection,” Sci. Rep. 6(1), 19419 (2016).
[Crossref] [PubMed]

d’Amora, M.

I. Coto Hernández, M. Castello, L. Lanzanò, M. d’Amora, P. Bianchini, A. Diaspro, and G. Vicidomini, “Two-Photon Excitation STED Microscopy with Time-Gated Detection,” Sci. Rep. 6(1), 19419 (2016).
[Crossref] [PubMed]

Deguchi, T.

M. Castello, G. Tortarolo, I. Coto Hernández, T. Deguchi, A. Diaspro, and G. Vicidomini, “Removal of anti-Stokes emission background in STED microscopy by FPGA-based synchronous detection,” Rev. Sci. Instrum. 88(5), 053701 (2017).
[Crossref] [PubMed]

Diaspro, A.

M. Castello, G. Tortarolo, I. Coto Hernández, T. Deguchi, A. Diaspro, and G. Vicidomini, “Removal of anti-Stokes emission background in STED microscopy by FPGA-based synchronous detection,” Rev. Sci. Instrum. 88(5), 053701 (2017).
[Crossref] [PubMed]

I. Coto Hernández, M. Castello, L. Lanzanò, M. d’Amora, P. Bianchini, A. Diaspro, and G. Vicidomini, “Two-Photon Excitation STED Microscopy with Time-Gated Detection,” Sci. Rep. 6(1), 19419 (2016).
[Crossref] [PubMed]

M. Castello, G. Tortarolo, I. C. Hernández, P. Bianchini, M. Buttafava, G. Boso, A. Tosi, A. Diaspro, and G. Vicidomini, “Gated-sted microscopy with subnanosecond pulsed fiber laser for reducing photobleaching,” Microsc. Res. Tech. 79(9), 785–791 (2016).
[Crossref] [PubMed]

P. Bianchini, B. Harke, S. Galiani, G. Vicidomini, and A. Diaspro, “Single-wavelength two-photon excitation-stimulated emission depletion (SW2PE-STED) superresolution imaging,” Proc. Natl. Acad. Sci. U.S.A. 109(17), 6390–6393 (2012).
[Crossref] [PubMed]

Ding, J. B.

K. T. Takasaki, J. B. Ding, and B. L. Sabatini, “Live-Cell Superresolution Imaging by Pulsed STED Two-Photon Excitation Microscopy,” Biophys. J. 104(4), 770–777 (2013).
[Crossref] [PubMed]

Dresbach, T.

Eggeling, C.

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]

Engelhardt, J.

Fang, Y.-C.

K. Otomo, T. Hibi, Y.-C. Fang, J.-H. Hung, M. Tsutsumi, R. Kawakami, H. Yokoyama, and T. Nemoto, “Advanced easySTED microscopy based on two-photon excitation by electrical modulations of light pulse wavefronts,” Biomed. Opt. Express 9(6), 2671–2680 (2018).
[Crossref] [PubMed]

K. Sawada, R. Kawakami, Y.-C. Fang, J.-H. Hung, Y. Kozawa, K. Otomo, S. Sato, H. Yokoyama, and T. Nemoto, “Improvement of two-photon microscopic imaging in deep regions of living mouse brains by utilizing a light source based on an electrically controllable gain-switched laser diode,” Proc. SPIE 10498, 104982L (2018).
[Crossref]

J.-H. Hung, K. Sato, Y.-C. Fang, L.-H. Peng, T. Nemoto, and H. Yokoyama, “Generation of high-peak-power sub-nanosecond 650-nm-band optical pulses based on semiconductor-laser-controlling technologies,” Appl. Phys. Express 10(10), 102701 (2017).
[Crossref]

R. Kawakami, K. Sawada, Y. Kusama, Y.-C. Fang, S. Kanazawa, Y. Kozawa, S. Sato, H. Yokoyama, and T. Nemoto, “In vivo two-photon imaging of mouse hippocampal neurons in dentate gyrus using a light source based on a high-peak power gain-switched laser diode,” Biomed. Opt. Express 6(3), 891–901 (2015).
[Crossref] [PubMed]

Galiani, S.

P. Bianchini, B. Harke, S. Galiani, G. Vicidomini, and A. Diaspro, “Single-wavelength two-photon excitation-stimulated emission depletion (SW2PE-STED) superresolution imaging,” Proc. Natl. Acad. Sci. U.S.A. 109(17), 6390–6393 (2012).
[Crossref] [PubMed]

Garner, C. C.

R. G. Zhai, H. Vardinon-Friedman, C. Cases-Langhoff, B. Becker, E. D. Gundelfinger, N. E. Ziv, and C. C. Garner, “Assembling the Presynaptic Active Zone: A Characterization of an Active One Precursor Vesicle,” Neuron 29(1), 131–143 (2001).
[Crossref] [PubMed]

Ghelani, T.

T. Ghelani and S. J. Sigrist, “Coupling the Structural and Functional Assembly of Synaptic Release Sites,” Front. Neuroanat. 12, 81 (2018).
[Crossref] [PubMed]

Gundelfinger, E. D.

R. G. Zhai, H. Vardinon-Friedman, C. Cases-Langhoff, B. Becker, E. D. Gundelfinger, N. E. Ziv, and C. C. Garner, “Assembling the Presynaptic Active Zone: A Characterization of an Active One Precursor Vesicle,” Neuron 29(1), 131–143 (2001).
[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]

Harke, B.

P. Bianchini, B. Harke, S. Galiani, G. Vicidomini, and A. Diaspro, “Single-wavelength two-photon excitation-stimulated emission depletion (SW2PE-STED) superresolution imaging,” Proc. Natl. Acad. Sci. U.S.A. 109(17), 6390–6393 (2012).
[Crossref] [PubMed]

Hashimoto, N.

Heintzmann, R.

L. Schermelleh, R. Heintzmann, and H. Leonhardt, “A guide to super-resolution fluorescence microscopy,” J. Cell Biol. 190(2), 165–175 (2010).
[Crossref] [PubMed]

Hell, S. W.

J. Oracz, V. Westphal, C. Radzewicz, S. J. Sahl, and S. W. Hell, “Photobleaching in STED nanoscopy and its dependence on the photon flux applied for reversible silencing of the fluorophore,” Sci. Rep. 7(1), 11354 (2017).
[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]

S. Schrof, T. Staudt, E. Rittweger, N. Wittenmayer, T. Dresbach, J. Engelhardt, and S. W. Hell, “STED nanoscopy with mass-produced laser diodes,” Opt. Express 19(9), 8066–8072 (2011).
[Crossref] [PubMed]

G. Moneron and S. W. Hell, “Two-photon excitation STED microscopy,” Opt. Express 17(17), 14567–14573 (2009).
[Crossref] [PubMed]

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

Hernández, I. C.

M. Castello, G. Tortarolo, I. C. Hernández, P. Bianchini, M. Buttafava, G. Boso, A. Tosi, A. Diaspro, and G. Vicidomini, “Gated-sted microscopy with subnanosecond pulsed fiber laser for reducing photobleaching,” Microsc. Res. Tech. 79(9), 785–791 (2016).
[Crossref] [PubMed]

Hibi, T.

Hung, J.-H.

K. Sawada, R. Kawakami, Y.-C. Fang, J.-H. Hung, Y. Kozawa, K. Otomo, S. Sato, H. Yokoyama, and T. Nemoto, “Improvement of two-photon microscopic imaging in deep regions of living mouse brains by utilizing a light source based on an electrically controllable gain-switched laser diode,” Proc. SPIE 10498, 104982L (2018).
[Crossref]

K. Otomo, T. Hibi, Y.-C. Fang, J.-H. Hung, M. Tsutsumi, R. Kawakami, H. Yokoyama, and T. Nemoto, “Advanced easySTED microscopy based on two-photon excitation by electrical modulations of light pulse wavefronts,” Biomed. Opt. Express 9(6), 2671–2680 (2018).
[Crossref] [PubMed]

J.-H. Hung, K. Sato, Y.-C. Fang, L.-H. Peng, T. Nemoto, and H. Yokoyama, “Generation of high-peak-power sub-nanosecond 650-nm-band optical pulses based on semiconductor-laser-controlling technologies,” Appl. Phys. Express 10(10), 102701 (2017).
[Crossref]

Ingaramo, M.

Kanazawa, S.

Kawakami, R.

Kozawa, Y.

Kurihara, M.

Kusama, Y.

Lanzanò, L.

I. Coto Hernández, M. Castello, L. Lanzanò, M. d’Amora, P. Bianchini, A. Diaspro, and G. Vicidomini, “Two-Photon Excitation STED Microscopy with Time-Gated Detection,” Sci. Rep. 6(1), 19419 (2016).
[Crossref] [PubMed]

Leonhardt, H.

L. Schermelleh, R. Heintzmann, and H. Leonhardt, “A guide to super-resolution fluorescence microscopy,” J. Cell Biol. 190(2), 165–175 (2010).
[Crossref] [PubMed]

Li, Q.

Q. Li, S. S. H. Wu, and K. C. Chou, “Subdiffraction-limit two-photon fluorescence microscopy for GFP-tagged cell imaging,” Biophys. J. 97(12), 3224–3228 (2009).
[Crossref] [PubMed]

Marsicano, G.

P. Bethge, R. Chéreau, E. Avignone, G. Marsicano, and U. V. Nägerl, “Two-photon excitation STED microscopy in two colors in acute brain slices,” Biophys. J. 104(4), 778–785 (2013).
[Crossref] [PubMed]

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]

G. Moneron and S. W. Hell, “Two-photon excitation STED microscopy,” Opt. Express 17(17), 14567–14573 (2009).
[Crossref] [PubMed]

Nägerl, U. V.

P. Bethge, R. Chéreau, E. Avignone, G. Marsicano, and U. V. Nägerl, “Two-photon excitation STED microscopy in two colors in acute brain slices,” Biophys. J. 104(4), 778–785 (2013).
[Crossref] [PubMed]

Nemoto, T.

K. Sawada, R. Kawakami, Y.-C. Fang, J.-H. Hung, Y. Kozawa, K. Otomo, S. Sato, H. Yokoyama, and T. Nemoto, “Improvement of two-photon microscopic imaging in deep regions of living mouse brains by utilizing a light source based on an electrically controllable gain-switched laser diode,” Proc. SPIE 10498, 104982L (2018).
[Crossref]

K. Otomo, T. Hibi, Y.-C. Fang, J.-H. Hung, M. Tsutsumi, R. Kawakami, H. Yokoyama, and T. Nemoto, “Advanced easySTED microscopy based on two-photon excitation by electrical modulations of light pulse wavefronts,” Biomed. Opt. Express 9(6), 2671–2680 (2018).
[Crossref] [PubMed]

J.-H. Hung, K. Sato, Y.-C. Fang, L.-H. Peng, T. Nemoto, and H. Yokoyama, “Generation of high-peak-power sub-nanosecond 650-nm-band optical pulses based on semiconductor-laser-controlling technologies,” Appl. Phys. Express 10(10), 102701 (2017).
[Crossref]

R. Kawakami, K. Sawada, Y. Kusama, Y.-C. Fang, S. Kanazawa, Y. Kozawa, S. Sato, H. Yokoyama, and T. Nemoto, “In vivo two-photon imaging of mouse hippocampal neurons in dentate gyrus using a light source based on a high-peak power gain-switched laser diode,” Biomed. Opt. Express 6(3), 891–901 (2015).
[Crossref] [PubMed]

K. Otomo, T. Hibi, Y. Kozawa, M. Kurihara, N. Hashimoto, H. Yokoyama, S. Sato, and T. Nemoto, “Two-photon excitation STED microscopy by utilizing transmissive liquid crystal devices,” Opt. Express 22(23), 28215–28221 (2014).
[Crossref] [PubMed]

Y. Kusama, Y. Tanushi, M. Yokoyama, R. Kawakami, T. Hibi, Y. Kozawa, T. Nemoto, S. Sato, and H. Yokoyama, “7-ps optical pulse generation from a 1064-nm gain-switched laser diode and its application for two-photon microscopy,” Opt. Express 22(5), 5746–5753 (2014).
[Crossref] [PubMed]

R. Kawakami, K. Sawada, A. Sato, T. Hibi, Y. Kozawa, S. Sato, H. Yokoyama, and T. Nemoto, “Visualizing hippocampal neurons with in vivo two-photon microscopy using a 1030 nm picosecond pulse laser,” Sci. Rep. 3(1), 1014 (2013).
[Crossref] [PubMed]

Nogare, D. D.

Oracz, J.

J. Oracz, V. Westphal, C. Radzewicz, S. J. Sahl, and S. W. Hell, “Photobleaching in STED nanoscopy and its dependence on the photon flux applied for reversible silencing of the fluorophore,” Sci. Rep. 7(1), 11354 (2017).
[Crossref] [PubMed]

Otomo, K.

Patterson, G. H.

Peng, L.-H.

J.-H. Hung, K. Sato, Y.-C. Fang, L.-H. Peng, T. Nemoto, and H. Yokoyama, “Generation of high-peak-power sub-nanosecond 650-nm-band optical pulses based on semiconductor-laser-controlling technologies,” Appl. Phys. Express 10(10), 102701 (2017).
[Crossref]

Radzewicz, C.

J. Oracz, V. Westphal, C. Radzewicz, S. J. Sahl, and S. W. Hell, “Photobleaching in STED nanoscopy and its dependence on the photon flux applied for reversible silencing of the fluorophore,” Sci. Rep. 7(1), 11354 (2017).
[Crossref] [PubMed]

Rittweger, E.

Sabatini, B. L.

K. T. Takasaki, J. B. Ding, and B. L. Sabatini, “Live-Cell Superresolution Imaging by Pulsed STED Two-Photon Excitation Microscopy,” Biophys. J. 104(4), 770–777 (2013).
[Crossref] [PubMed]

Sahl, S. J.

J. Oracz, V. Westphal, C. Radzewicz, S. J. Sahl, and S. W. Hell, “Photobleaching in STED nanoscopy and its dependence on the photon flux applied for reversible silencing of the fluorophore,” Sci. Rep. 7(1), 11354 (2017).
[Crossref] [PubMed]

Sato, A.

R. Kawakami, K. Sawada, A. Sato, T. Hibi, Y. Kozawa, S. Sato, H. Yokoyama, and T. Nemoto, “Visualizing hippocampal neurons with in vivo two-photon microscopy using a 1030 nm picosecond pulse laser,” Sci. Rep. 3(1), 1014 (2013).
[Crossref] [PubMed]

Sato, K.

J.-H. Hung, K. Sato, Y.-C. Fang, L.-H. Peng, T. Nemoto, and H. Yokoyama, “Generation of high-peak-power sub-nanosecond 650-nm-band optical pulses based on semiconductor-laser-controlling technologies,” Appl. Phys. Express 10(10), 102701 (2017).
[Crossref]

Sato, S.

Sawada, K.

K. Sawada, R. Kawakami, Y.-C. Fang, J.-H. Hung, Y. Kozawa, K. Otomo, S. Sato, H. Yokoyama, and T. Nemoto, “Improvement of two-photon microscopic imaging in deep regions of living mouse brains by utilizing a light source based on an electrically controllable gain-switched laser diode,” Proc. SPIE 10498, 104982L (2018).
[Crossref]

R. Kawakami, K. Sawada, Y. Kusama, Y.-C. Fang, S. Kanazawa, Y. Kozawa, S. Sato, H. Yokoyama, and T. Nemoto, “In vivo two-photon imaging of mouse hippocampal neurons in dentate gyrus using a light source based on a high-peak power gain-switched laser diode,” Biomed. Opt. Express 6(3), 891–901 (2015).
[Crossref] [PubMed]

R. Kawakami, K. Sawada, A. Sato, T. Hibi, Y. Kozawa, S. Sato, H. Yokoyama, and T. Nemoto, “Visualizing hippocampal neurons with in vivo two-photon microscopy using a 1030 nm picosecond pulse laser,” Sci. Rep. 3(1), 1014 (2013).
[Crossref] [PubMed]

Schermelleh, L.

L. Schermelleh, R. Heintzmann, and H. Leonhardt, “A guide to super-resolution fluorescence microscopy,” J. Cell Biol. 190(2), 165–175 (2010).
[Crossref] [PubMed]

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]

Schrof, S.

Shroff, H.

Sigrist, S. J.

T. Ghelani and S. J. Sigrist, “Coupling the Structural and Functional Assembly of Synaptic Release Sites,” Front. Neuroanat. 12, 81 (2018).
[Crossref] [PubMed]

Staudt, T.

Svoboda, K.

K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron 50(6), 823–839 (2006).
[Crossref] [PubMed]

Ta, H.

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]

Takasaki, K. T.

K. T. Takasaki, J. B. Ding, and B. L. Sabatini, “Live-Cell Superresolution Imaging by Pulsed STED Two-Photon Excitation Microscopy,” Biophys. J. 104(4), 770–777 (2013).
[Crossref] [PubMed]

Tanushi, Y.

Tortarolo, G.

M. Castello, G. Tortarolo, I. Coto Hernández, T. Deguchi, A. Diaspro, and G. Vicidomini, “Removal of anti-Stokes emission background in STED microscopy by FPGA-based synchronous detection,” Rev. Sci. Instrum. 88(5), 053701 (2017).
[Crossref] [PubMed]

M. Castello, G. Tortarolo, I. C. Hernández, P. Bianchini, M. Buttafava, G. Boso, A. Tosi, A. Diaspro, and G. Vicidomini, “Gated-sted microscopy with subnanosecond pulsed fiber laser for reducing photobleaching,” Microsc. Res. Tech. 79(9), 785–791 (2016).
[Crossref] [PubMed]

Tosi, A.

M. Castello, G. Tortarolo, I. C. Hernández, P. Bianchini, M. Buttafava, G. Boso, A. Tosi, A. Diaspro, and G. Vicidomini, “Gated-sted microscopy with subnanosecond pulsed fiber laser for reducing photobleaching,” Microsc. Res. Tech. 79(9), 785–791 (2016).
[Crossref] [PubMed]

Tsutsumi, M.

Vardinon-Friedman, H.

R. G. Zhai, H. Vardinon-Friedman, C. Cases-Langhoff, B. Becker, E. D. Gundelfinger, N. E. Ziv, and C. C. Garner, “Assembling the Presynaptic Active Zone: A Characterization of an Active One Precursor Vesicle,” Neuron 29(1), 131–143 (2001).
[Crossref] [PubMed]

Vicidomini, G.

M. Castello, G. Tortarolo, I. Coto Hernández, T. Deguchi, A. Diaspro, and G. Vicidomini, “Removal of anti-Stokes emission background in STED microscopy by FPGA-based synchronous detection,” Rev. Sci. Instrum. 88(5), 053701 (2017).
[Crossref] [PubMed]

I. Coto Hernández, M. Castello, L. Lanzanò, M. d’Amora, P. Bianchini, A. Diaspro, and G. Vicidomini, “Two-Photon Excitation STED Microscopy with Time-Gated Detection,” Sci. Rep. 6(1), 19419 (2016).
[Crossref] [PubMed]

M. Castello, G. Tortarolo, I. C. Hernández, P. Bianchini, M. Buttafava, G. Boso, A. Tosi, A. Diaspro, and G. Vicidomini, “Gated-sted microscopy with subnanosecond pulsed fiber laser for reducing photobleaching,” Microsc. Res. Tech. 79(9), 785–791 (2016).
[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]

P. Bianchini, B. Harke, S. Galiani, G. Vicidomini, and A. Diaspro, “Single-wavelength two-photon excitation-stimulated emission depletion (SW2PE-STED) superresolution imaging,” Proc. Natl. Acad. Sci. U.S.A. 109(17), 6390–6393 (2012).
[Crossref] [PubMed]

Webb, W. W.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[Crossref] [PubMed]

Westphal, V.

J. Oracz, V. Westphal, C. Radzewicz, S. J. Sahl, and S. W. Hell, “Photobleaching in STED nanoscopy and its dependence on the photon flux applied for reversible silencing of the fluorophore,” Sci. Rep. 7(1), 11354 (2017).
[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).

Williams, R. M.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[Crossref] [PubMed]

Winter, P. W.

Wittenmayer, N.

Wu, S. S. H.

Q. Li, S. S. H. Wu, and K. C. Chou, “Subdiffraction-limit two-photon fluorescence microscopy for GFP-tagged cell imaging,” Biophys. J. 97(12), 3224–3228 (2009).
[Crossref] [PubMed]

Yasuda, R.

K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron 50(6), 823–839 (2006).
[Crossref] [PubMed]

Yokoyama, H.

K. Sawada, R. Kawakami, Y.-C. Fang, J.-H. Hung, Y. Kozawa, K. Otomo, S. Sato, H. Yokoyama, and T. Nemoto, “Improvement of two-photon microscopic imaging in deep regions of living mouse brains by utilizing a light source based on an electrically controllable gain-switched laser diode,” Proc. SPIE 10498, 104982L (2018).
[Crossref]

K. Otomo, T. Hibi, Y.-C. Fang, J.-H. Hung, M. Tsutsumi, R. Kawakami, H. Yokoyama, and T. Nemoto, “Advanced easySTED microscopy based on two-photon excitation by electrical modulations of light pulse wavefronts,” Biomed. Opt. Express 9(6), 2671–2680 (2018).
[Crossref] [PubMed]

J.-H. Hung, K. Sato, Y.-C. Fang, L.-H. Peng, T. Nemoto, and H. Yokoyama, “Generation of high-peak-power sub-nanosecond 650-nm-band optical pulses based on semiconductor-laser-controlling technologies,” Appl. Phys. Express 10(10), 102701 (2017).
[Crossref]

R. Kawakami, K. Sawada, Y. Kusama, Y.-C. Fang, S. Kanazawa, Y. Kozawa, S. Sato, H. Yokoyama, and T. Nemoto, “In vivo two-photon imaging of mouse hippocampal neurons in dentate gyrus using a light source based on a high-peak power gain-switched laser diode,” Biomed. Opt. Express 6(3), 891–901 (2015).
[Crossref] [PubMed]

K. Otomo, T. Hibi, Y. Kozawa, M. Kurihara, N. Hashimoto, H. Yokoyama, S. Sato, and T. Nemoto, “Two-photon excitation STED microscopy by utilizing transmissive liquid crystal devices,” Opt. Express 22(23), 28215–28221 (2014).
[Crossref] [PubMed]

Y. Kusama, Y. Tanushi, M. Yokoyama, R. Kawakami, T. Hibi, Y. Kozawa, T. Nemoto, S. Sato, and H. Yokoyama, “7-ps optical pulse generation from a 1064-nm gain-switched laser diode and its application for two-photon microscopy,” Opt. Express 22(5), 5746–5753 (2014).
[Crossref] [PubMed]

R. Kawakami, K. Sawada, A. Sato, T. Hibi, Y. Kozawa, S. Sato, H. Yokoyama, and T. Nemoto, “Visualizing hippocampal neurons with in vivo two-photon microscopy using a 1030 nm picosecond pulse laser,” Sci. Rep. 3(1), 1014 (2013).
[Crossref] [PubMed]

Yokoyama, M.

York, A. G.

Zhai, R. G.

R. G. Zhai, H. Vardinon-Friedman, C. Cases-Langhoff, B. Becker, E. D. Gundelfinger, N. E. Ziv, and C. C. Garner, “Assembling the Presynaptic Active Zone: A Characterization of an Active One Precursor Vesicle,” Neuron 29(1), 131–143 (2001).
[Crossref] [PubMed]

Zipfel, W. R.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[Crossref] [PubMed]

Ziv, N. E.

R. G. Zhai, H. Vardinon-Friedman, C. Cases-Langhoff, B. Becker, E. D. Gundelfinger, N. E. Ziv, and C. C. Garner, “Assembling the Presynaptic Active Zone: A Characterization of an Active One Precursor Vesicle,” Neuron 29(1), 131–143 (2001).
[Crossref] [PubMed]

Appl. Phys. Express (1)

J.-H. Hung, K. Sato, Y.-C. Fang, L.-H. Peng, T. Nemoto, and H. Yokoyama, “Generation of high-peak-power sub-nanosecond 650-nm-band optical pulses based on semiconductor-laser-controlling technologies,” Appl. Phys. Express 10(10), 102701 (2017).
[Crossref]

Biomed. Opt. Express (2)

Biophys. J. (3)

Q. Li, S. S. H. Wu, and K. C. Chou, “Subdiffraction-limit two-photon fluorescence microscopy for GFP-tagged cell imaging,” Biophys. J. 97(12), 3224–3228 (2009).
[Crossref] [PubMed]

K. T. Takasaki, J. B. Ding, and B. L. Sabatini, “Live-Cell Superresolution Imaging by Pulsed STED Two-Photon Excitation Microscopy,” Biophys. J. 104(4), 770–777 (2013).
[Crossref] [PubMed]

P. Bethge, R. Chéreau, E. Avignone, G. Marsicano, and U. V. Nägerl, “Two-photon excitation STED microscopy in two colors in acute brain slices,” Biophys. J. 104(4), 778–785 (2013).
[Crossref] [PubMed]

Front. Neuroanat. (1)

T. Ghelani and S. J. Sigrist, “Coupling the Structural and Functional Assembly of Synaptic Release Sites,” Front. Neuroanat. 12, 81 (2018).
[Crossref] [PubMed]

J. Cell Biol. (1)

L. Schermelleh, R. Heintzmann, and H. Leonhardt, “A guide to super-resolution fluorescence microscopy,” J. Cell Biol. 190(2), 165–175 (2010).
[Crossref] [PubMed]

Microsc. Res. Tech. (1)

M. Castello, G. Tortarolo, I. C. Hernández, P. Bianchini, M. Buttafava, G. Boso, A. Tosi, A. Diaspro, and G. Vicidomini, “Gated-sted microscopy with subnanosecond pulsed fiber laser for reducing photobleaching,” Microsc. Res. Tech. 79(9), 785–791 (2016).
[Crossref] [PubMed]

Nat. Biotechnol. (1)

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003).
[Crossref] [PubMed]

Neuron (2)

K. Svoboda and R. Yasuda, “Principles of two-photon excitation microscopy and its applications to neuroscience,” Neuron 50(6), 823–839 (2006).
[Crossref] [PubMed]

R. G. Zhai, H. Vardinon-Friedman, C. Cases-Langhoff, B. Becker, E. D. Gundelfinger, N. E. Ziv, and C. C. Garner, “Assembling the Presynaptic Active Zone: A Characterization of an Active One Precursor Vesicle,” Neuron 29(1), 131–143 (2001).
[Crossref] [PubMed]

Opt. Express (4)

Optica (1)

Phys. Rev. Lett. (1)

V. Westphal and S. W. Hell, “Nanoscale Resolution in the Focal Plane of an Optical Microscope,” Phys. Rev. Lett. 94(14), 143903 (2005).

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. (1)

P. Bianchini, B. Harke, S. Galiani, G. Vicidomini, and A. Diaspro, “Single-wavelength two-photon excitation-stimulated emission depletion (SW2PE-STED) superresolution imaging,” Proc. Natl. Acad. Sci. U.S.A. 109(17), 6390–6393 (2012).
[Crossref] [PubMed]

Proc. SPIE (1)

K. Sawada, R. Kawakami, Y.-C. Fang, J.-H. Hung, Y. Kozawa, K. Otomo, S. Sato, H. Yokoyama, and T. Nemoto, “Improvement of two-photon microscopic imaging in deep regions of living mouse brains by utilizing a light source based on an electrically controllable gain-switched laser diode,” Proc. SPIE 10498, 104982L (2018).
[Crossref]

Rev. Sci. Instrum. (1)

M. Castello, G. Tortarolo, I. Coto Hernández, T. Deguchi, A. Diaspro, and G. Vicidomini, “Removal of anti-Stokes emission background in STED microscopy by FPGA-based synchronous detection,” Rev. Sci. Instrum. 88(5), 053701 (2017).
[Crossref] [PubMed]

Sci. Rep. (3)

R. Kawakami, K. Sawada, A. Sato, T. Hibi, Y. Kozawa, S. Sato, H. Yokoyama, and T. Nemoto, “Visualizing hippocampal neurons with in vivo two-photon microscopy using a 1030 nm picosecond pulse laser,” Sci. Rep. 3(1), 1014 (2013).
[Crossref] [PubMed]

I. Coto Hernández, M. Castello, L. Lanzanò, M. d’Amora, P. Bianchini, A. Diaspro, and G. Vicidomini, “Two-Photon Excitation STED Microscopy with Time-Gated Detection,” Sci. Rep. 6(1), 19419 (2016).
[Crossref] [PubMed]

J. Oracz, V. Westphal, C. Radzewicz, S. J. Sahl, and S. W. Hell, “Photobleaching in STED nanoscopy and its dependence on the photon flux applied for reversible silencing of the fluorophore,” Sci. Rep. 7(1), 11354 (2017).
[Crossref] [PubMed]

Science (1)

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

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Figures (7)

Fig. 1
Fig. 1 Overview of the optical setup (DM: dichroic mirror; GaAsP NDD: gallium arsenide phosphide-based non-descanned detector; HWP: half-wave plate; LD: laser diode; M: mirror; tLCDs: transmissive liquid crystal devices).
Fig. 2
Fig. 2 Optical waveform (a, c) and spectrum (b, d) of 655-nm STED pulse (a, b) and 638-nm-LD STED pulse (c, d).
Fig. 3
Fig. 3 Comparison of fluorescence depletion efficiencies of 655-nm and 638-nm-LD STED beams for 1-µm Nile red beads: (a) fluorescent images of 1-µm Nile red bead under three irradiation conditions; (b) fluorescence intensity profiles along the dashed lines of (a); (c) dependence of fluorescence depletion efficiencies on average power of each STED beam at focal plane for 1-µm Nile red bead.
Fig. 4
Fig. 4 Fluorescence depletion efficiencies of 655-nm and 638-nm-LD STED beams against fluorescent probes frequently used in bioimaging (asterisks indicate negative fluorescence depletion efficiency percentage; error bars represent standard deviation (n = 3)).
Fig. 5
Fig. 5 Fluorescent image of 100-nm red bead excited by doughnut-shaped 655-nm STED beam; the dashed line shows the focal plane.
Fig. 6
Fig. 6 Comparison of TPLSM and TP-tLC-STED images. (a) TPLSM and TP-tLC-STED images of 20-nm Nile red bead. Lower graphs show fluorescence intensities around the intensity centers along the x- and y-axes for the corresponding upper images. Blue lines indicate the fitted Gaussian curves of the measurement values (gray dots). (b) Dependence of FWHM values of 20-nm Nile red bead images on 655-nm STED power at the focal plane. The FWHM values along x-axis (red) and y-axis (green) were fitted with the inverse square root function obtained by the employed equation [20]. (c) Scan number dependency of peak fluorescence intensity of 20-nm Nile red bead images under 3.3 mW, 655-nm STED light beam irradiation. (d) Comparison of TPLSM and TP-tLC-STED images of COS-7 cell immunostained with anti-α-tubulin followed by the AlexaFluor546-conjugated secondary antibody. The fluorescent intensity profiles along the dashed lines are shown in the graph.
Fig. 7
Fig. 7 (a) TPLSM images of cultured neuronal cell immunostained with anti-Bassoon followed by ATTO532-conjugated secondary antibody. (b) Comparison of TPLSM and TP-tLC-STED images of the area marked with white squares in (a). (c) Fluorescent intensity profiles along dashed lines.