Abstract

In this paper, we report the enhancement of resolution of continuous wave (CW) stimulated emission depletion (STED) microscopy by a novel method of structured illumination of an excitation beam. Illumination by multiple excitation beams through the specific pupil apertures with high in-plane wave vectors leads to interference of diffracted light flux near the focal plane, resulting in the contraction of the point spread function (PSF) of the excitation. Light spot reduction by the suggested standing wave (SW) illumination method contributes to make up much lower depletion efficiency of the CW STED microscopy than that of the pulsed STED method. First, theoretical analysis showed that the full width at half maximum (FWHM) of the effective PSF on the detection plane is expected to be smaller than 25% of that of conventional CW STED. Second, through the simulation, it was elucidated that both the donut-shaped PSF of the depletion beam and the confocal optics suppress undesired contribution of sidelobes of the PSF by the SW illumination to the effective PSF of the STED system. Finally, through the imaging experiment on 40-nm fluorescent beads with the developed SW-CW STED microscopy system, we obtained the result which follows the overall tendency from the simulation in the aspects of resolution improvement and reduction of sidelobes. Based on the obtained result, we expect that the proposed method can become one of the strategies to enhance the resolution of the CW STED microscopy.

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

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2017 (1)

2016 (2)

2015 (2)

B. Neupane, T. Jin, L. F. Mellor, E. G. Loboa, F. S. Ligler, and G. Wang, “Continuous-Wave Stimulated Emission Depletion Microscope for Imaging Actin Cytoskeleton in Fixed and Live Cells,” Sensors (Basel) 15(9), 24178–24190 (2015).
[Crossref] [PubMed]

M. Saxena, G. Eluru, and S. S. Gorthi, “Structured illumination microscopy,” Adv. Opt. Photonics 7(2), 241 (2015).
[Crossref]

2014 (2)

G. Vicidomini, I. C. Hernández, M. d’Amora, F. C. Zanacchi, P. Bianchini, and A. Diaspro, “Gated CW-STED microscopy: A versatile tool for biological nanometer scale investigation,” Methods 66(2), 124–130 (2014).
[Crossref] [PubMed]

S. Beater, P. Holzmeister, E. Pibiri, B. Lalkens, and P. Tinnefeld, “Choosing dyes for cw-STED nanoscopy using self-assembled nanorulers,” Phys. Chem. Chem. Phys. 16(15), 6990–6996 (2014).
[Crossref] [PubMed]

2013 (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]

2011 (2)

J. R. Moffitt, C. Osseforth, and J. Michaelis, “Time-gating improves the spatial resolution of STED microscopy,” Opt. Express 19(5), 4242–4254 (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 (2)

2009 (2)

O. Gliko, W. E. Brownell, and P. Saggau, “Fast two-dimensional standing-wave total-internal-reflection fluorescence microscopy using acousto-optic deflectors,” Opt. Lett. 34(6), 836–838 (2009).
[Crossref] [PubMed]

J. Lu, W. Min, J.-A. Conchello, X. S. Xie, and J. W. Lichtman, “Super-Resolution Laser Scanning Microscopy through Spatiotemporal Modulation,” Nano Lett. 9(11), 3883–3889 (2009).
[Crossref] [PubMed]

2008 (2)

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]

2007 (2)

E. Chung, D. Kim, Y. Cui, Y.-H. Kim, and P. T. C. So, “Two-dimensional standing wave total internal reflection fluorescence microscopy: superresolution imaging of single molecular and biological specimens,” Biophys. J. 93(5), 1747–1757 (2007).
[Crossref] [PubMed]

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[Crossref] [PubMed]

2006 (1)

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]

1994 (1)

1992 (1)

1959 (2)

E. Wolf, “Electromagnetic Diffraction in Optical Systems. I. An Integral Representation of the Image Field,” Proc. R. Soc. A Math. Phys. Eng. Sci. 253, 349–357 (1959).

B. Richards and E. Wolf, “Electromagnetic Diffraction in Optical Systems. II. Structure of the Image Field in an Aplanatic System,” Proc. R. Soc. A Math. Phys. Eng. Sci. 253, 358–379 (1959).

Beater, S.

S. Beater, P. Holzmeister, E. Pibiri, B. Lalkens, and P. Tinnefeld, “Choosing dyes for cw-STED nanoscopy using self-assembled nanorulers,” Phys. Chem. Chem. Phys. 16(15), 6990–6996 (2014).
[Crossref] [PubMed]

Bianchini, P.

G. Vicidomini, I. C. Hernández, M. d’Amora, F. C. Zanacchi, P. Bianchini, and A. Diaspro, “Gated CW-STED microscopy: A versatile tool for biological nanometer scale investigation,” Methods 66(2), 124–130 (2014).
[Crossref] [PubMed]

Boruah, B. R.

Brownell, W. E.

Chung, E.

E. Chung, D. Kim, Y. Cui, Y.-H. Kim, and P. T. C. So, “Two-dimensional standing wave total internal reflection fluorescence microscopy: superresolution imaging of single molecular and biological specimens,” Biophys. J. 93(5), 1747–1757 (2007).
[Crossref] [PubMed]

E. Chung, D. Kim, and P. T. So, “Extended resolution wide-field optical imaging: objective-launched standing-wave total internal reflection fluorescence microscopy,” Opt. Lett. 31(7), 945–947 (2006).
[Crossref] [PubMed]

Conchello, J.-A.

J. Lu, W. Min, J.-A. Conchello, X. S. Xie, and J. W. Lichtman, “Super-Resolution Laser Scanning Microscopy through Spatiotemporal Modulation,” Nano Lett. 9(11), 3883–3889 (2009).
[Crossref] [PubMed]

Cui, Y.

E. Chung, D. Kim, Y. Cui, Y.-H. Kim, and P. T. C. So, “Two-dimensional standing wave total internal reflection fluorescence microscopy: superresolution imaging of single molecular and biological specimens,” Biophys. J. 93(5), 1747–1757 (2007).
[Crossref] [PubMed]

d’Amora, M.

G. Vicidomini, I. C. Hernández, M. d’Amora, F. C. Zanacchi, P. Bianchini, and A. Diaspro, “Gated CW-STED microscopy: A versatile tool for biological nanometer scale investigation,” Methods 66(2), 124–130 (2014).
[Crossref] [PubMed]

Diaspro, A.

G. Vicidomini, I. C. Hernández, M. d’Amora, F. C. Zanacchi, P. Bianchini, and A. Diaspro, “Gated CW-STED microscopy: A versatile tool for biological nanometer scale investigation,” Methods 66(2), 124–130 (2014).
[Crossref] [PubMed]

Ding, X.

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, 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. 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]

Eluru, G.

M. Saxena, G. Eluru, and S. S. Gorthi, “Structured illumination microscopy,” Adv. Opt. Photonics 7(2), 241 (2015).
[Crossref]

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).
[Crossref] [PubMed]

Gao, P.

Gliko, O.

Gong, W.

Gorthi, S. S.

M. Saxena, G. Eluru, and S. S. Gorthi, “Structured illumination microscopy,” Adv. Opt. Photonics 7(2), 241 (2015).
[Crossref]

Guo, Q.

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]

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]

Harke, B.

B. Harke, C. K. Ullal, J. Keller, and S. W. Hell, “Three-Dimensional Nanoscopy of Colloidal Crystals,” Nano Lett. 8(5), 1309–1313 (2008).
[Crossref] [PubMed]

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]

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[Crossref] [PubMed]

Hell, S.

Hell, S. W.

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. 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]

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]

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (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.

G. Vicidomini, I. C. Hernández, M. d’Amora, F. C. Zanacchi, P. Bianchini, and A. Diaspro, “Gated CW-STED microscopy: A versatile tool for biological nanometer scale investigation,” Methods 66(2), 124–130 (2014).
[Crossref] [PubMed]

Holzmeister, P.

S. Beater, P. Holzmeister, E. Pibiri, B. Lalkens, and P. Tinnefeld, “Choosing dyes for cw-STED nanoscopy using self-assembled nanorulers,” Phys. Chem. Chem. Phys. 16(15), 6990–6996 (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]

Jin, T.

B. Neupane, T. Jin, L. F. Mellor, E. G. Loboa, F. S. Ligler, and G. Wang, “Continuous-Wave Stimulated Emission Depletion Microscope for Imaging Actin Cytoskeleton in Fixed and Live Cells,” Sensors (Basel) 15(9), 24178–24190 (2015).
[Crossref] [PubMed]

Keller, J.

B. Harke, C. K. Ullal, J. Keller, and S. W. Hell, “Three-Dimensional Nanoscopy of Colloidal Crystals,” Nano Lett. 8(5), 1309–1313 (2008).
[Crossref] [PubMed]

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]

Kim, D.

E. Chung, D. Kim, Y. Cui, Y.-H. Kim, and P. T. C. So, “Two-dimensional standing wave total internal reflection fluorescence microscopy: superresolution imaging of single molecular and biological specimens,” Biophys. J. 93(5), 1747–1757 (2007).
[Crossref] [PubMed]

E. Chung, D. Kim, and P. T. So, “Extended resolution wide-field optical imaging: objective-launched standing-wave total internal reflection fluorescence microscopy,” Opt. Lett. 31(7), 945–947 (2006).
[Crossref] [PubMed]

Kim, Y.-H.

E. Chung, D. Kim, Y. Cui, Y.-H. Kim, and P. T. C. So, “Two-dimensional standing wave total internal reflection fluorescence microscopy: superresolution imaging of single molecular and biological specimens,” Biophys. J. 93(5), 1747–1757 (2007).
[Crossref] [PubMed]

Klar, T. A.

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

Lalkens, B.

S. Beater, P. Holzmeister, E. Pibiri, B. Lalkens, and P. Tinnefeld, “Choosing dyes for cw-STED nanoscopy using self-assembled nanorulers,” Phys. Chem. Chem. Phys. 16(15), 6990–6996 (2014).
[Crossref] [PubMed]

Leutenegger, M.

Lichtman, J. W.

J. Lu, W. Min, J.-A. Conchello, X. S. Xie, and J. W. Lichtman, “Super-Resolution Laser Scanning Microscopy through Spatiotemporal Modulation,” Nano Lett. 9(11), 3883–3889 (2009).
[Crossref] [PubMed]

Ligler, F. S.

B. Neupane, T. Jin, L. F. Mellor, E. G. Loboa, F. S. Ligler, and G. Wang, “Continuous-Wave Stimulated Emission Depletion Microscope for Imaging Actin Cytoskeleton in Fixed and Live Cells,” Sensors (Basel) 15(9), 24178–24190 (2015).
[Crossref] [PubMed]

Loboa, E. G.

B. Neupane, T. Jin, L. F. Mellor, E. G. Loboa, F. S. Ligler, and G. Wang, “Continuous-Wave Stimulated Emission Depletion Microscope for Imaging Actin Cytoskeleton in Fixed and Live Cells,” Sensors (Basel) 15(9), 24178–24190 (2015).
[Crossref] [PubMed]

Lu, J.

J. Lu, W. Min, J.-A. Conchello, X. S. Xie, and J. W. Lichtman, “Super-Resolution Laser Scanning Microscopy through Spatiotemporal Modulation,” Nano Lett. 9(11), 3883–3889 (2009).
[Crossref] [PubMed]

Medda, R.

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[Crossref] [PubMed]

Mellor, L. F.

B. Neupane, T. Jin, L. F. Mellor, E. G. Loboa, F. S. Ligler, and G. Wang, “Continuous-Wave Stimulated Emission Depletion Microscope for Imaging Actin Cytoskeleton in Fixed and Live Cells,” Sensors (Basel) 15(9), 24178–24190 (2015).
[Crossref] [PubMed]

Michaelis, J.

Min, W.

J. Lu, W. Min, J.-A. Conchello, X. S. Xie, and J. W. Lichtman, “Super-Resolution Laser Scanning Microscopy through Spatiotemporal Modulation,” Nano Lett. 9(11), 3883–3889 (2009).
[Crossref] [PubMed]

Moffitt, J. R.

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. 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]

Neupane, B.

B. Neupane, T. Jin, L. F. Mellor, E. G. Loboa, F. S. Ligler, and G. Wang, “Continuous-Wave Stimulated Emission Depletion Microscope for Imaging Actin Cytoskeleton in Fixed and Live Cells,” Sensors (Basel) 15(9), 24178–24190 (2015).
[Crossref] [PubMed]

Ni, H.

Nienhaus, G. U.

Osseforth, C.

Pibiri, E.

S. Beater, P. Holzmeister, E. Pibiri, B. Lalkens, and P. Tinnefeld, “Choosing dyes for cw-STED nanoscopy using self-assembled nanorulers,” Phys. Chem. Chem. Phys. 16(15), 6990–6996 (2014).
[Crossref] [PubMed]

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]

Richards, B.

B. Richards and E. Wolf, “Electromagnetic Diffraction in Optical Systems. II. Structure of the Image Field in an Aplanatic System,” Proc. R. Soc. A Math. Phys. Eng. Sci. 253, 358–379 (1959).

Saggau, P.

Saxena, M.

M. Saxena, G. Eluru, and S. S. Gorthi, “Structured illumination microscopy,” Adv. Opt. Photonics 7(2), 241 (2015).
[Crossref]

Schönle, A.

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

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

Shen, S.

Si, K.

So, P. T.

So, P. T. C.

E. Chung, D. Kim, Y. Cui, Y.-H. Kim, and P. T. C. So, “Two-dimensional standing wave total internal reflection fluorescence microscopy: superresolution imaging of single molecular and biological specimens,” Biophys. J. 93(5), 1747–1757 (2007).
[Crossref] [PubMed]

Stelzer, E. H. K.

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]

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]

Tinnefeld, P.

S. Beater, P. Holzmeister, E. Pibiri, B. Lalkens, and P. Tinnefeld, “Choosing dyes for cw-STED nanoscopy using self-assembled nanorulers,” Phys. Chem. Chem. Phys. 16(15), 6990–6996 (2014).
[Crossref] [PubMed]

Ullal, C. K.

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]

Vicidomini, G.

G. Vicidomini, I. C. Hernández, M. d’Amora, F. C. Zanacchi, P. Bianchini, and A. Diaspro, “Gated CW-STED microscopy: A versatile tool for biological nanometer scale investigation,” Methods 66(2), 124–130 (2014).
[Crossref] [PubMed]

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

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]

Wang, G.

B. Neupane, T. Jin, L. F. Mellor, E. G. Loboa, F. S. Ligler, and G. Wang, “Continuous-Wave Stimulated Emission Depletion Microscope for Imaging Actin Cytoskeleton in Fixed and Live Cells,” Sensors (Basel) 15(9), 24178–24190 (2015).
[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]

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

Wichmann, J.

Willig, K. I.

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[Crossref] [PubMed]

Wolf, E.

B. Richards and E. Wolf, “Electromagnetic Diffraction in Optical Systems. II. Structure of the Image Field in an Aplanatic System,” Proc. R. Soc. A Math. Phys. Eng. Sci. 253, 358–379 (1959).

E. Wolf, “Electromagnetic Diffraction in Optical Systems. I. An Integral Representation of the Image Field,” Proc. R. Soc. A Math. Phys. Eng. Sci. 253, 349–357 (1959).

Xie, X. S.

J. Lu, W. Min, J.-A. Conchello, X. S. Xie, and J. W. Lichtman, “Super-Resolution Laser Scanning Microscopy through Spatiotemporal Modulation,” Nano Lett. 9(11), 3883–3889 (2009).
[Crossref] [PubMed]

Zanacchi, F. C.

G. Vicidomini, I. C. Hernández, M. d’Amora, F. C. Zanacchi, P. Bianchini, and A. Diaspro, “Gated CW-STED microscopy: A versatile tool for biological nanometer scale investigation,” Methods 66(2), 124–130 (2014).
[Crossref] [PubMed]

Zheng, Y.

Zhu, B.

Zou, L.

Adv. Opt. Photonics (1)

M. Saxena, G. Eluru, and S. S. Gorthi, “Structured illumination microscopy,” Adv. Opt. Photonics 7(2), 241 (2015).
[Crossref]

Appl. Opt. (1)

Biophys. J. (1)

E. Chung, D. Kim, Y. Cui, Y.-H. Kim, and P. T. C. So, “Two-dimensional standing wave total internal reflection fluorescence microscopy: superresolution imaging of single molecular and biological specimens,” Biophys. J. 93(5), 1747–1757 (2007).
[Crossref] [PubMed]

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

Methods (1)

G. Vicidomini, I. C. Hernández, M. d’Amora, F. C. Zanacchi, P. Bianchini, and A. Diaspro, “Gated CW-STED microscopy: A versatile tool for biological nanometer scale investigation,” Methods 66(2), 124–130 (2014).
[Crossref] [PubMed]

Nano Lett. (2)

J. Lu, W. Min, J.-A. Conchello, X. S. Xie, and J. W. Lichtman, “Super-Resolution Laser Scanning Microscopy through Spatiotemporal Modulation,” Nano Lett. 9(11), 3883–3889 (2009).
[Crossref] [PubMed]

B. Harke, C. K. Ullal, J. Keller, and S. W. Hell, “Three-Dimensional Nanoscopy of Colloidal Crystals,” Nano Lett. 8(5), 1309–1313 (2008).
[Crossref] [PubMed]

Nat. Methods (2)

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]

K. I. Willig, B. Harke, R. Medda, and S. W. Hell, “STED microscopy with continuous wave beams,” Nat. Methods 4(11), 915–918 (2007).
[Crossref] [PubMed]

Opt. Express (5)

Opt. Lett. (4)

Phys. Chem. Chem. Phys. (1)

S. Beater, P. Holzmeister, E. Pibiri, B. Lalkens, and P. Tinnefeld, “Choosing dyes for cw-STED nanoscopy using self-assembled nanorulers,” Phys. Chem. Chem. Phys. 16(15), 6990–6996 (2014).
[Crossref] [PubMed]

PLoS One (1)

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

Proc. Natl. Acad. Sci. U.S.A. (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. R. Soc. A Math. Phys. Eng. Sci. (2)

E. Wolf, “Electromagnetic Diffraction in Optical Systems. I. An Integral Representation of the Image Field,” Proc. R. Soc. A Math. Phys. Eng. Sci. 253, 349–357 (1959).

B. Richards and E. Wolf, “Electromagnetic Diffraction in Optical Systems. II. Structure of the Image Field in an Aplanatic System,” Proc. R. Soc. A Math. Phys. Eng. Sci. 253, 358–379 (1959).

Sensors (Basel) (1)

B. Neupane, T. Jin, L. F. Mellor, E. G. Loboa, F. S. Ligler, and G. Wang, “Continuous-Wave Stimulated Emission Depletion Microscope for Imaging Actin Cytoskeleton in Fixed and Live Cells,” Sensors (Basel) 15(9), 24178–24190 (2015).
[Crossref] [PubMed]

Other (1)

F. Zijp, “Near-Field Optical Data Storage, Ph.D. Dissertation,” (Ph.D. Dissertation, Delft University of Technology, 2007).

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Figures (17)

Fig. 1
Fig. 1 Schematics of conventional and SW-CW STED microscopies. The excitation PSF of the SW-CW STED microscopy is spatially modulated by the interference of high-aperture-angle illumination.
Fig. 2
Fig. 2 Optical scheme for the calculation of electric field vector near the focal plane of the aplanatic optics.
Fig. 3
Fig. 3 Examples of the apertures in the entrance pupil. Light flux incident to the area of blocking aperture is blocked by it. In this paper, the aperture shown in (a) is referred as dipole-shaped aperture, and aperture shown in (b) is referred as central flux blocking aperture.
Fig. 4
Fig. 4 Normalized PSF in the image plane on the surface of the measurement sample for the various cases with different values of ρ in the configuration of the dipole shaped aperture as shown in Fig. 3(a). (a) and (b) show the PSFs in the x- and y-directions induced by illumination of excitation beam only. (c) and (d) show the effective emission PSFs in the x- and y-directions induced by depletion beam.
Fig. 5
Fig. 5 Normalized PSF in the image plane on the surface of the measurement sample for the various cases with different values of ρ in the configuration of the central flux blocking aperture as shown in Fig. 3(b). (a) and (b) show the PSFs in the x- and y-directions induced by illumination of excitation beam only. (c) and (d) show the effective emission PSFs in the x- and y-directions induced by depletion beam.
Fig. 6
Fig. 6 Normalized PSF at the image plane on the detector for the various cases with different value of ρ in the configuration of the central flux blocking aperture as shown in Fig. 3(b). (a) and (b) show the PSFs in the x-direction for confocal imaging without STED effect and with STED imaging, respectively. (c) and (d) show the effective PSFs in the y-direction for confocal imaging without STED effect and with STED imaging, respectively.
Fig. 7
Fig. 7 Comparison of the FWHM of the PSF at the detection plane in x- and y-directions depending on the blocking ratio ρ for the cases with central flux blocking aperture with the conventional CW STED. The right y-axis shows the ratio of the height of the sidelobe to the height of the main-lobe.
Fig. 8
Fig. 8 Optical layout of the SW-CW STED microscopy. Sample is held to a 3-axis piezo stage for scanning. FS – femtosecond; M – mirror; FI – Faraday isolator; HWP – half-wave plate; GTP – Glan-Thompson polarizer; L – lens; SC – super-continuum device; F – band pass filter; FM – flip mirror; PMF – polarization maintaining fiber; VPP – vortex phase plate; QWP – quarter-wave plate; DM – dichroic mirror; A – aperture; OL – objective lens; TL – tube lens; MMF – multi-mode fiber; APD – avalanche photodiode.
Fig. 9
Fig. 9 Conceptual diagram of the central blocking aperture (a) and the fabricated mask containing various apertures with different blocking ratio ρ (b). The diameter of the collimated light beam incident to each aperture is 5 mm. Hence, for each given blocking ratio the size of the blocking aperture can be determined. For example, for the blocking ratio of 0.5, blocking dimension, t, in x-direction is 2.5 mm.
Fig. 10
Fig. 10 Comparison of each focal spot images of 80 nm gold nano-beads in xy-planes. (a) and (b) are, respectively, the images of obtained with conventional confocal (excitation beam with circular polarization) and depletion donut beam. (c)-(f) show images by the proposed SW excitation beam with different blocking ratio of 0.4, 0.5, 0.6, and 0.7, respectively. Scale bars correspond to 1µm Each image was normalized for comparable intensity conditions. (g) shows the spot profiles of each spots.
Fig. 11
Fig. 11 Fluorescence images with yellow-green fluorescent beads with 40 nm diameter for (a1-a3) conventional CW STED microscopy and (b1-b4, c1-c2) SW-CW STED microscopy with respect to different depletion power condition from 0 to 300 mW. Especially, the scanned image area of (a1-a3) and (b1-b4) is the same. The scale bars correspond to 2 μm.
Fig. 12
Fig. 12 FWHM values of SW-CW STED and conventional CW STED. (a) Simulation results for depletion beam power of 0 to 3 W. (b) Average FWHM values of conventional cases and SW cases with respect to depletion power shown in each images of Fig. 11, which are compared with the simulation results.
Fig. 13
Fig. 13 (a) Comparison of fluorescence images of conventional CW STED microscopy and SW-CW STED microscopy for depletion power of 0 mW and 300 mW. (b) Profiles in the x and y directions of the spot pointed by white arrows in each images of (a).
Fig. 14
Fig. 14 Considered Types of Apertures.
Fig. 15
Fig. 15 For the simulation model shown in Fig. 14(a), simulated PSFs in the x-direction of (a) the excitation beam only on the sample plane, (b) depleted emission on the sample plane, (c) confocal system without STED effect on the detection plane, and (d) SW STED on the detection plane. In the figure, only “Central Block ρ = 0.5 y-axis” PSF in y-direction for comparison.
Fig. 16
Fig. 16 For the simulation model shown in Fig. 14(b), simulated PSFs in the x-direction of (a) the excitation beam only on the sample plane, (b) depleted emission on the sample plane, (c) confocal system without STED effect on the detection plane, and (d) SW-CW STED on the detection plane. In the figure, only “Central Block ρ = 0.5 y-axis” PSF in y-direction for comparison.
Fig. 17
Fig. 17 Comparison of focal spot images of (a) SW confocal with blocking ratio ρ 0.7, (b) conventional confocal, and (c) donut-shaped depletion beam in xz-planes with 80 nm gold nano-beads. (d) Intensity distribution profile of each confocal spots along the axial direction. The scale bars correspond to 1 μm.

Tables (2)

Tables Icon

Table 1 Comparison of FWHM values of experimental results in Fig. 10 and simulation results.

Tables Icon

Table 2 List of FWHM of PSFs in the x-direction on several observation plane considering various conditions of illumination and types of apertures. Only central flux blocking aperture shows values of FWHM for both x and y directions.

Equations (5)

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E( x p , y p , z p )= i 2π Ω E 1 ( k x , k y ) k z e i( k x x p + k y y p + k z z p ) d k x d k y ,
E 1 ( k x , k y )= P 1 RP A m ( k x , k y ),
[ | k x |kNA( ρ 0 +Δρ ) ] 2 + k y 2 [ ΔρkNA ] 2 ,
| k x |ρkNA .
I SW,conf = I ill ( I obj P pinhole )

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