Abstract

We developed a compact stimulated emission depletion (STED) two-photon excitation microscopy that utilized electrically controllable components. Transmissive liquid crystal devices inserted directly in front of the objective lens converted the STED light into an optical vortex while leaving the excitation light unaffected. Light pulses of two different colors, 1.06 and 0.64 μm, were generated by laser diode-based light sources, and the delay between the two pulses was flexibly controlled so as to maximize the fluorescence suppression ratio. In our experiments, the spatial resolution of this system was up to three times higher than that obtained without STED light irradiation, and we successfully visualize the fine microtubule network structures in fixed mammalian cells without causing significant photo-damage.

© 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement

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  1. W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
    [Crossref] [PubMed]
  2. 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]
  3. 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]
  4. S. W. Hell, “Far-field optical nanoscopy,” Science 316(5828), 1153–1158 (2007).
    [Crossref] [PubMed]
  5. B. Huang, H. Babcock, and X. Zhuang, “Breaking the diffraction barrier: super-resolution imaging of cells,” Cell 143(7), 1047–1058 (2010).
    [Crossref] [PubMed]
  6. 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]
  7. 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]
  8. G. Moneron and S. W. Hell, “Two-photon excitation STED microscopy,” Opt. Express 17(17), 14567–14573 (2009).
    [Crossref] [PubMed]
  9. 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]
  10. 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]
  11. M. Reuss, J. Engelhardt, and S. W. Hell, “Birefringent device converts a standard scanning microscope into a STED microscope that also maps molecular orientation,” Opt. Express 18(2), 1049–1058 (2010).
    [Crossref] [PubMed]
  12. F. Görlitz, P. Hoyer, H. J. Falk, L. Kastrup, J. Engelhardt, and S. W. Hell, “A STED microscope designed for routine biomedical applications,” Prog. Electromagnetics Res. 147, 57–68 (2014).
    [Crossref]
  13. 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]
  14. S. Ipponjima, T. Hibi, Y. Kozawa, H. Horanai, H. Yokoyama, S. Sato, and T. Nemoto, “Improvement of lateral resolution and extension of depth of field in two-photon microscopy by a higher-order radially polarized beam,” Microscopy (Oxf.) 63(1), 23–32 (2014).
    [Crossref] [PubMed]
  15. N. Hashimoto and M. Kurihara, “Liquid crystal quantized GRIN lens and its application to AF systems,” Proc. SPIE 7232, 808123 (2009).
    [Crossref]
  16. A. K. Dutta, K. Kamada, and K. Ohta, “Spectroscopic studies of nile red in organic solvents and polymers,” J. Photochem. Photobiol. Chem. 93(1), 57–64 (1996).
    [Crossref]
  17. M. Booth, D. Andrade, D. Burke, B. Patton, and M. Zurauskas, “Aberrations and adaptive optics in super-resolution microscopy,” Microscopy (Oxf.) 64(4), 251–261 (2015).
    [Crossref] [PubMed]
  18. T. J. Gould, D. Burke, J. Bewersdorf, and M. J. Booth, “Adaptive optics enables 3D STED microscopy in aberrating specimens,” Opt. Express 20(19), 20998–21009 (2012).
    [Crossref] [PubMed]
  19. A. Tanabe, T. Hibi, S. Ipponjima, K. Matsumoto, M. Yokoyama, M. Kurihara, N. Hashimoto, and T. Nemoto, “Correcting spherical aberrations in a biospecimen using a transmissive liquid crystal device in two-photon excitation laser scanning microscopy,” J. Biomed. Opt. 20(10), 101204 (2015).
    [Crossref] [PubMed]
  20. A. Tanabe, T. Hibi, S. Ipponjima, K. Matsumoto, M. Yokoyama, M. Kurihara, N. Hashimoto, and T. Nemoto, “Transmissive Liquid-crystal device for correcting primary coma aberration and astigmatism in biospecimen in two-photon excitation laser scanning microscopy,” J. Biomed. Opt. 21(12), 121503 (2016).
    [Crossref] [PubMed]
  21. M. Leutenegger, C. Eggeling, and S. W. Hell, “Analytical description of STED microscopy performance,” Opt. Express 18(25), 26417–26429 (2010).
    [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. J.-H. Hung, K. Sato, Y.-C. Fang, L.-T. 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]
  24. 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]
  25. I. C. 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]

2017 (1)

J.-H. Hung, K. Sato, Y.-C. Fang, L.-T. 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. C. 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]

A. Tanabe, T. Hibi, S. Ipponjima, K. Matsumoto, M. Yokoyama, M. Kurihara, N. Hashimoto, and T. Nemoto, “Transmissive Liquid-crystal device for correcting primary coma aberration and astigmatism in biospecimen in two-photon excitation laser scanning microscopy,” J. Biomed. Opt. 21(12), 121503 (2016).
[Crossref] [PubMed]

2015 (3)

M. Booth, D. Andrade, D. Burke, B. Patton, and M. Zurauskas, “Aberrations and adaptive optics in super-resolution microscopy,” Microscopy (Oxf.) 64(4), 251–261 (2015).
[Crossref] [PubMed]

A. Tanabe, T. Hibi, S. Ipponjima, K. Matsumoto, M. Yokoyama, M. Kurihara, N. Hashimoto, and T. Nemoto, “Correcting spherical aberrations in a biospecimen using a transmissive liquid crystal device in two-photon excitation laser scanning microscopy,” J. Biomed. Opt. 20(10), 101204 (2015).
[Crossref] [PubMed]

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]

2014 (4)

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]

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]

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

S. Ipponjima, T. Hibi, Y. Kozawa, H. Horanai, H. Yokoyama, S. Sato, and T. Nemoto, “Improvement of lateral resolution and extension of depth of field in two-photon microscopy by a higher-order radially polarized beam,” Microscopy (Oxf.) 63(1), 23–32 (2014).
[Crossref] [PubMed]

2013 (2)

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)

2011 (2)

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. 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]

2010 (3)

2009 (2)

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

N. Hashimoto and M. Kurihara, “Liquid crystal quantized GRIN lens and its application to AF systems,” Proc. SPIE 7232, 808123 (2009).
[Crossref]

2007 (1)

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

2000 (1)

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

1996 (1)

A. K. Dutta, K. Kamada, and K. Ohta, “Spectroscopic studies of nile red in organic solvents and polymers,” J. Photochem. Photobiol. Chem. 93(1), 57–64 (1996).
[Crossref]

1994 (1)

1990 (1)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Andrade, D.

M. Booth, D. Andrade, D. Burke, B. Patton, and M. Zurauskas, “Aberrations and adaptive optics in super-resolution microscopy,” Microscopy (Oxf.) 64(4), 251–261 (2015).
[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]

Babcock, H.

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

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]

Bewersdorf, J.

Bianchini, P.

I. C. 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]

Booth, M.

M. Booth, D. Andrade, D. Burke, B. Patton, and M. Zurauskas, “Aberrations and adaptive optics in super-resolution microscopy,” Microscopy (Oxf.) 64(4), 251–261 (2015).
[Crossref] [PubMed]

Booth, M. J.

Burke, D.

M. Booth, D. Andrade, D. Burke, B. Patton, and M. Zurauskas, “Aberrations and adaptive optics in super-resolution microscopy,” Microscopy (Oxf.) 64(4), 251–261 (2015).
[Crossref] [PubMed]

T. J. Gould, D. Burke, J. Bewersdorf, and M. J. Booth, “Adaptive optics enables 3D STED microscopy in aberrating specimens,” Opt. Express 20(19), 20998–21009 (2012).
[Crossref] [PubMed]

Castello, M.

I. C. 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]

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]

d’Amora, M.

I. C. 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]

Denk, W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Diaspro, A.

I. C. 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]

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.

Dutta, A. K.

A. K. Dutta, K. Kamada, and K. Ohta, “Spectroscopic studies of nile red in organic solvents and polymers,” J. Photochem. Photobiol. Chem. 93(1), 57–64 (1996).
[Crossref]

Dyba, M.

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

Eggeling, C.

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]

Egner, A.

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

Engelhardt, J.

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

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

Falk, H. J.

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

Fang, Y.-C.

J.-H. Hung, K. Sato, Y.-C. Fang, L.-T. 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]

Görlitz, F.

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

Gould, T. J.

Han, K. Y.

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]

Hashimoto, N.

A. Tanabe, T. Hibi, S. Ipponjima, K. Matsumoto, M. Yokoyama, M. Kurihara, N. Hashimoto, and T. Nemoto, “Transmissive Liquid-crystal device for correcting primary coma aberration and astigmatism in biospecimen in two-photon excitation laser scanning microscopy,” J. Biomed. Opt. 21(12), 121503 (2016).
[Crossref] [PubMed]

A. Tanabe, T. Hibi, S. Ipponjima, K. Matsumoto, M. Yokoyama, M. Kurihara, N. Hashimoto, and T. Nemoto, “Correcting spherical aberrations in a biospecimen using a transmissive liquid crystal device in two-photon excitation laser scanning microscopy,” J. Biomed. Opt. 20(10), 101204 (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]

N. Hashimoto and M. Kurihara, “Liquid crystal quantized GRIN lens and its application to AF systems,” Proc. SPIE 7232, 808123 (2009).
[Crossref]

Hell, S. W.

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

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. 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]

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

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]

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

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

Hernández, I. C.

I. C. 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]

Hibi, T.

A. Tanabe, T. Hibi, S. Ipponjima, K. Matsumoto, M. Yokoyama, M. Kurihara, N. Hashimoto, and T. Nemoto, “Transmissive Liquid-crystal device for correcting primary coma aberration and astigmatism in biospecimen in two-photon excitation laser scanning microscopy,” J. Biomed. Opt. 21(12), 121503 (2016).
[Crossref] [PubMed]

A. Tanabe, T. Hibi, S. Ipponjima, K. Matsumoto, M. Yokoyama, M. Kurihara, N. Hashimoto, and T. Nemoto, “Correcting spherical aberrations in a biospecimen using a transmissive liquid crystal device in two-photon excitation laser scanning microscopy,” J. Biomed. Opt. 20(10), 101204 (2015).
[Crossref] [PubMed]

S. Ipponjima, T. Hibi, Y. Kozawa, H. Horanai, H. Yokoyama, S. Sato, and T. Nemoto, “Improvement of lateral resolution and extension of depth of field in two-photon microscopy by a higher-order radially polarized beam,” Microscopy (Oxf.) 63(1), 23–32 (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]

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]

Horanai, H.

S. Ipponjima, T. Hibi, Y. Kozawa, H. Horanai, H. Yokoyama, S. Sato, and T. Nemoto, “Improvement of lateral resolution and extension of depth of field in two-photon microscopy by a higher-order radially polarized beam,” Microscopy (Oxf.) 63(1), 23–32 (2014).
[Crossref] [PubMed]

Hoyer, P.

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

Huang, B.

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

Hung, J.-H.

J.-H. Hung, K. Sato, Y.-C. Fang, L.-T. 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]

Ipponjima, S.

A. Tanabe, T. Hibi, S. Ipponjima, K. Matsumoto, M. Yokoyama, M. Kurihara, N. Hashimoto, and T. Nemoto, “Transmissive Liquid-crystal device for correcting primary coma aberration and astigmatism in biospecimen in two-photon excitation laser scanning microscopy,” J. Biomed. Opt. 21(12), 121503 (2016).
[Crossref] [PubMed]

A. Tanabe, T. Hibi, S. Ipponjima, K. Matsumoto, M. Yokoyama, M. Kurihara, N. Hashimoto, and T. Nemoto, “Correcting spherical aberrations in a biospecimen using a transmissive liquid crystal device in two-photon excitation laser scanning microscopy,” J. Biomed. Opt. 20(10), 101204 (2015).
[Crossref] [PubMed]

S. Ipponjima, T. Hibi, Y. Kozawa, H. Horanai, H. Yokoyama, S. Sato, and T. Nemoto, “Improvement of lateral resolution and extension of depth of field in two-photon microscopy by a higher-order radially polarized beam,” Microscopy (Oxf.) 63(1), 23–32 (2014).
[Crossref] [PubMed]

Jakobs, S.

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]

Kamada, K.

A. K. Dutta, K. Kamada, and K. Ohta, “Spectroscopic studies of nile red in organic solvents and polymers,” J. Photochem. Photobiol. Chem. 93(1), 57–64 (1996).
[Crossref]

Kanazawa, S.

Kastrup, L.

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

Kawakami, R.

Klar, T. A.

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

Kozawa, Y.

Kurihara, M.

A. Tanabe, T. Hibi, S. Ipponjima, K. Matsumoto, M. Yokoyama, M. Kurihara, N. Hashimoto, and T. Nemoto, “Transmissive Liquid-crystal device for correcting primary coma aberration and astigmatism in biospecimen in two-photon excitation laser scanning microscopy,” J. Biomed. Opt. 21(12), 121503 (2016).
[Crossref] [PubMed]

A. Tanabe, T. Hibi, S. Ipponjima, K. Matsumoto, M. Yokoyama, M. Kurihara, N. Hashimoto, and T. Nemoto, “Correcting spherical aberrations in a biospecimen using a transmissive liquid crystal device in two-photon excitation laser scanning microscopy,” J. Biomed. Opt. 20(10), 101204 (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]

N. Hashimoto and M. Kurihara, “Liquid crystal quantized GRIN lens and its application to AF systems,” Proc. SPIE 7232, 808123 (2009).
[Crossref]

Kusama, Y.

Lanzanò, L.

I. C. 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]

Leutenegger, M.

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]

Matsumoto, K.

A. Tanabe, T. Hibi, S. Ipponjima, K. Matsumoto, M. Yokoyama, M. Kurihara, N. Hashimoto, and T. Nemoto, “Transmissive Liquid-crystal device for correcting primary coma aberration and astigmatism in biospecimen in two-photon excitation laser scanning microscopy,” J. Biomed. Opt. 21(12), 121503 (2016).
[Crossref] [PubMed]

A. Tanabe, T. Hibi, S. Ipponjima, K. Matsumoto, M. Yokoyama, M. Kurihara, N. Hashimoto, and T. Nemoto, “Correcting spherical aberrations in a biospecimen using a transmissive liquid crystal device in two-photon excitation laser scanning microscopy,” J. Biomed. Opt. 20(10), 101204 (2015).
[Crossref] [PubMed]

Moneron, G.

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]

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.

J.-H. Hung, K. Sato, Y.-C. Fang, L.-T. 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]

A. Tanabe, T. Hibi, S. Ipponjima, K. Matsumoto, M. Yokoyama, M. Kurihara, N. Hashimoto, and T. Nemoto, “Transmissive Liquid-crystal device for correcting primary coma aberration and astigmatism in biospecimen in two-photon excitation laser scanning microscopy,” J. Biomed. Opt. 21(12), 121503 (2016).
[Crossref] [PubMed]

A. Tanabe, T. Hibi, S. Ipponjima, K. Matsumoto, M. Yokoyama, M. Kurihara, N. Hashimoto, and T. Nemoto, “Correcting spherical aberrations in a biospecimen using a transmissive liquid crystal device in two-photon excitation laser scanning microscopy,” J. Biomed. Opt. 20(10), 101204 (2015).
[Crossref] [PubMed]

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]

S. Ipponjima, T. Hibi, Y. Kozawa, H. Horanai, H. Yokoyama, S. Sato, and T. Nemoto, “Improvement of lateral resolution and extension of depth of field in two-photon microscopy by a higher-order radially polarized beam,” Microscopy (Oxf.) 63(1), 23–32 (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]

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]

Ohta, K.

A. K. Dutta, K. Kamada, and K. Ohta, “Spectroscopic studies of nile red in organic solvents and polymers,” J. Photochem. Photobiol. Chem. 93(1), 57–64 (1996).
[Crossref]

Otomo, K.

Patton, B.

M. Booth, D. Andrade, D. Burke, B. Patton, and M. Zurauskas, “Aberrations and adaptive optics in super-resolution microscopy,” Microscopy (Oxf.) 64(4), 251–261 (2015).
[Crossref] [PubMed]

Peng, L.-T.

J.-H. Hung, K. Sato, Y.-C. Fang, L.-T. 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]

Reuss, M.

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

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]

Sato, K.

J.-H. Hung, K. Sato, Y.-C. Fang, L.-T. 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.

Schrof, S.

Staudt, T.

Strickler, J. H.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Ta, H.

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]

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]

Tanabe, A.

A. Tanabe, T. Hibi, S. Ipponjima, K. Matsumoto, M. Yokoyama, M. Kurihara, N. Hashimoto, and T. Nemoto, “Transmissive Liquid-crystal device for correcting primary coma aberration and astigmatism in biospecimen in two-photon excitation laser scanning microscopy,” J. Biomed. Opt. 21(12), 121503 (2016).
[Crossref] [PubMed]

A. Tanabe, T. Hibi, S. Ipponjima, K. Matsumoto, M. Yokoyama, M. Kurihara, N. Hashimoto, and T. Nemoto, “Correcting spherical aberrations in a biospecimen using a transmissive liquid crystal device in two-photon excitation laser scanning microscopy,” J. Biomed. Opt. 20(10), 101204 (2015).
[Crossref] [PubMed]

Tanushi, Y.

Vicidomini, G.

I. C. 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]

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]

Webb, W. W.

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

Westphal, V.

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]

Wichmann, J.

Wittenmayer, N.

Yokoyama, H.

Yokoyama, M.

A. Tanabe, T. Hibi, S. Ipponjima, K. Matsumoto, M. Yokoyama, M. Kurihara, N. Hashimoto, and T. Nemoto, “Transmissive Liquid-crystal device for correcting primary coma aberration and astigmatism in biospecimen in two-photon excitation laser scanning microscopy,” J. Biomed. Opt. 21(12), 121503 (2016).
[Crossref] [PubMed]

A. Tanabe, T. Hibi, S. Ipponjima, K. Matsumoto, M. Yokoyama, M. Kurihara, N. Hashimoto, and T. Nemoto, “Correcting spherical aberrations in a biospecimen using a transmissive liquid crystal device in two-photon excitation laser scanning microscopy,” J. Biomed. Opt. 20(10), 101204 (2015).
[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]

Zhuang, X.

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

Zurauskas, M.

M. Booth, D. Andrade, D. Burke, B. Patton, and M. Zurauskas, “Aberrations and adaptive optics in super-resolution microscopy,” Microscopy (Oxf.) 64(4), 251–261 (2015).
[Crossref] [PubMed]

Appl. Phys. Express (1)

J.-H. Hung, K. Sato, Y.-C. Fang, L.-T. 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 (1)

Biophys. J. (2)

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]

Cell (1)

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

J. Biomed. Opt. (2)

A. Tanabe, T. Hibi, S. Ipponjima, K. Matsumoto, M. Yokoyama, M. Kurihara, N. Hashimoto, and T. Nemoto, “Correcting spherical aberrations in a biospecimen using a transmissive liquid crystal device in two-photon excitation laser scanning microscopy,” J. Biomed. Opt. 20(10), 101204 (2015).
[Crossref] [PubMed]

A. Tanabe, T. Hibi, S. Ipponjima, K. Matsumoto, M. Yokoyama, M. Kurihara, N. Hashimoto, and T. Nemoto, “Transmissive Liquid-crystal device for correcting primary coma aberration and astigmatism in biospecimen in two-photon excitation laser scanning microscopy,” J. Biomed. Opt. 21(12), 121503 (2016).
[Crossref] [PubMed]

J. Photochem. Photobiol. Chem. (1)

A. K. Dutta, K. Kamada, and K. Ohta, “Spectroscopic studies of nile red in organic solvents and polymers,” J. Photochem. Photobiol. Chem. 93(1), 57–64 (1996).
[Crossref]

Microscopy (Oxf.) (2)

M. Booth, D. Andrade, D. Burke, B. Patton, and M. Zurauskas, “Aberrations and adaptive optics in super-resolution microscopy,” Microscopy (Oxf.) 64(4), 251–261 (2015).
[Crossref] [PubMed]

S. Ipponjima, T. Hibi, Y. Kozawa, H. Horanai, H. Yokoyama, S. Sato, and T. Nemoto, “Improvement of lateral resolution and extension of depth of field in two-photon microscopy by a higher-order radially polarized beam,” Microscopy (Oxf.) 63(1), 23–32 (2014).
[Crossref] [PubMed]

Nat. Methods (1)

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]

Opt. Express (7)

Opt. Lett. (1)

Proc. Natl. Acad. Sci. U.S.A. (1)

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

Proc. SPIE (1)

N. Hashimoto and M. Kurihara, “Liquid crystal quantized GRIN lens and its application to AF systems,” Proc. SPIE 7232, 808123 (2009).
[Crossref]

Prog. Electromagnetics Res. (1)

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

Sci. Rep. (1)

I. C. 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]

Science (2)

W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248(4951), 73–76 (1990).
[Crossref] [PubMed]

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 two-photon excitation (TP ex.) and STED light sources. DFB: distributed feedback; GS: gain-switched semiconductor; LD: laser diode.
Fig. 2
Fig. 2 (a) Schematic of our advanced easySTED-TP microscopy system. DM: dichroic mirror; GaAsP NDD: gallium arsenide phosphide-based non-descanned detector; GSLD: gain-switched laser diode; tLCD-G: transmissive liquid crystal device-based gradient index lens; tLCD-P; plain cell tLCD; tLCD-V: tLCD used to create optical vortices; λ/2: half-wave plate. (b) Polarization directions of the TP ex. and STED light beams and orientations of LC molecules in three different types of tLCDs. (c) Concentric transparent electrode pattern and phase distribution profile for a tLCD-G. (d) Theoretical phase distribution of the optical vortex generated by a tLCD-V.
Fig. 3
Fig. 3 Optical properties of the STED light pulse. (a) Optical spectrum. (b) Optical pulse shape.
Fig. 4
Fig. 4 Reconstructed fluorescent images of a 100 nm Red bead, excited by both the STED and two-photon excitation lights. (a) STED light, minimum voltage applied to the tLCD-G. (b) STED light, maximum voltage applied to the tLCD-G. (c) Two-photon excitation light.
Fig. 5
Fig. 5 (a) Fluorescent images of a 1 μm Nile Red bead under four different laser irradiation conditions. (b) Fluorescence intensity profiles around the intensity center and along the x-axis of (a). (c) Dependence of fluorescence intensity on STED power for a 1 μm Nile Red bead with and without two-photon excitation. (d) Dependence of the fluorescence intensity of a 1 μm Nile Red bead on the STED light pulse delay time (τdelay). The red dashed line indicates the fluorescence intensity obtained with only NIR light irradiation.
Fig. 6
Fig. 6 Comparison of TPLSM and advanced easySTED-TP images of 20 nm Nile Red beads. The upper panels show fluorescent images, while center and lower panels depict the fluorescence intensities around the intensity centers along the x- and y-axes for the corresponding upper-hand image. The red circles and blue lines indicate the measured values and fitted curves, respectively. The inset length values indicate the FWHM values.
Fig. 7
Fig. 7 (a) TPLSM image of microtubule networks in a fixed HeLa cell after immunostaining with ATTO 532-conjugated antibodies, reconstructed from z-stacks. (b) Comparison of TPLSM and advanced easySTED-TP images of basal (1.5 μm below the coverslip) and apical (9.0 μm below the coverslip) regions of (a). The inset panels indicate the fluorescence intensity profiles along the red dashed lines in the fluorescent images.