Abstract

We demonstrate that the lateral resolution of confocal laser scanning microscopy is dramatically improved by a higher-order radially polarized (HRP) beam with six concentric rings. This beam was generated simply by inserting liquid crystal devices in front of an objective lens. An HRP beam visualized aggregated 0.17 μm beads individually and is also applicable to biological imaging. This method can extend the capability of conventional laser scanning microscopes without modification of the system, with the exception of the addition of the liquid crystal devices in the optical path.

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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef] [PubMed]
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    [CrossRef]
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    [CrossRef] [PubMed]

2010 (2)

2009 (3)

2008 (2)

H. Wang, L. Shi, B. Lukyanchuk, C. Sheppard, and C. T. Chong, “Creation of a needle of longitudinally polarized light in vacuum using binary optics,” Nat. Photonics 2(8), 501–505 (2008).
[CrossRef]

B. Hein, K. I. Willig, and S. W. Hell, “Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell,” Proc. Natl. Acad. Sci. U.S.A. 105(38), 14271–14276 (2008).
[CrossRef] [PubMed]

2007 (1)

2006 (2)

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

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

2003 (2)

R. Dorn, S. Quabis, and G. Leuchs, “Sharper focus for a radially polarized light beam,” Phys. Rev. Lett. 91(23), 233901 (2003).
[CrossRef] [PubMed]

C. C. Sun and C. K. Liu, “Ultrasmall focusing spot with a long depth of focus based on polarization and phase modulation,” Opt. Lett. 28(2), 99–101 (2003).
[CrossRef] [PubMed]

2001 (1)

L. E. Helseth, “Roles of polarization, phase and amplitude in solid immersion lens systems,” Opt. Commun. 191(3-6), 161–172 (2001).
[CrossRef]

2000 (2)

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

S. Quabis, R. Dorn, M. Eberler, O. Glöckl, and G. Leuchs, “Focusing light to a tighter spot,” Opt. Commun. 179(1-6), 1–7 (2000).
[CrossRef]

1999 (1)

P. D. Higdon, P. Török, and T. Wilson, “Imaging properties of high aperture multiphoton fluorescence scanning optical microscopes,” J. Microsc. 193(2), 127–141 (1999).
[CrossRef]

1998 (2)

P. Török, P. D. Higdon, and T. Wilson, “Theory for confocal and conventional microscopes imaging small dielectric scatterers,” J. Mod. Opt. 45(8), 1681–1698 (1998).
[CrossRef]

A. A. Tovar, “Production and propagation of cylindrically polarized Laguerre-Gaussian laser beams,” J. Opt. Soc. Am. A 15(10), 2705–2711 (1998).
[CrossRef]

1997 (1)

C. J. R. Sheppard and P. Török, “An electromagnetic theory of imaging in fluorescence microscopy, and imaging in polarization fluorescence microscopy,” Bioimaging 5(4), 205–218 (1997).
[CrossRef]

1994 (1)

1987 (1)

1959 (1)

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A Math. Phys. Sci. 253(1274), 358–379 (1959).
[CrossRef]

Back, S. H.

J. Kim, D. C. Kim, and S. H. Back, “Demonstration of high lateral resolution in laser confocal microscopy using annular and radially polarized light,” Microsc. Res. Tech. 72(6), 441–446 (2009).
[CrossRef] [PubMed]

Bates, M.

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

Betzig, E.

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

Bonifacino, J. S.

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

Carlini, A. R.

Chong, C. T.

H. Wang, L. Shi, B. Lukyanchuk, C. Sheppard, and C. T. Chong, “Creation of a needle of longitudinally polarized light in vacuum using binary optics,” Nat. Photonics 2(8), 501–505 (2008).
[CrossRef]

Davidson, M. W.

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

De Koninck, Y.

Dehez, H.

Dorn, R.

R. Dorn, S. Quabis, and G. Leuchs, “Sharper focus for a radially polarized light beam,” Phys. Rev. Lett. 91(23), 233901 (2003).
[CrossRef] [PubMed]

S. Quabis, R. Dorn, M. Eberler, O. Glöckl, and G. Leuchs, “Focusing light to a tighter spot,” Opt. Commun. 179(1-6), 1–7 (2000).
[CrossRef]

Eberler, M.

S. Quabis, R. Dorn, M. Eberler, O. Glöckl, and G. Leuchs, “Focusing light to a tighter spot,” Opt. Commun. 179(1-6), 1–7 (2000).
[CrossRef]

Fontana, M. D.

Glöckl, O.

S. Quabis, R. Dorn, M. Eberler, O. Glöckl, and G. Leuchs, “Focusing light to a tighter spot,” Opt. Commun. 179(1-6), 1–7 (2000).
[CrossRef]

Gustafsson, M. G. L.

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

Hammoum, R.

Hein, B.

B. Hein, K. I. Willig, and S. W. Hell, “Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell,” Proc. Natl. Acad. Sci. U.S.A. 105(38), 14271–14276 (2008).
[CrossRef] [PubMed]

Hell, S. W.

B. Hein, K. I. Willig, and S. W. Hell, “Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell,” Proc. Natl. Acad. Sci. U.S.A. 105(38), 14271–14276 (2008).
[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]

Helseth, L. E.

L. E. Helseth, “Roles of polarization, phase and amplitude in solid immersion lens systems,” Opt. Commun. 191(3-6), 161–172 (2001).
[CrossRef]

Hess, H. F.

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

Higdon, P. D.

P. D. Higdon, P. Török, and T. Wilson, “Imaging properties of high aperture multiphoton fluorescence scanning optical microscopes,” J. Microsc. 193(2), 127–141 (1999).
[CrossRef]

P. Török, P. D. Higdon, and T. Wilson, “Theory for confocal and conventional microscopes imaging small dielectric scatterers,” J. Mod. Opt. 45(8), 1681–1698 (1998).
[CrossRef]

Kano, H.

Kim, D. C.

J. Kim, D. C. Kim, and S. H. Back, “Demonstration of high lateral resolution in laser confocal microscopy using annular and radially polarized light,” Microsc. Res. Tech. 72(6), 441–446 (2009).
[CrossRef] [PubMed]

Kim, J.

J. Kim, D. C. Kim, and S. H. Back, “Demonstration of high lateral resolution in laser confocal microscopy using annular and radially polarized light,” Microsc. Res. Tech. 72(6), 441–446 (2009).
[CrossRef] [PubMed]

Kozawa, Y.

Leuchs, G.

R. Dorn, S. Quabis, and G. Leuchs, “Sharper focus for a radially polarized light beam,” Phys. Rev. Lett. 91(23), 233901 (2003).
[CrossRef] [PubMed]

S. Quabis, R. Dorn, M. Eberler, O. Glöckl, and G. Leuchs, “Focusing light to a tighter spot,” Opt. Commun. 179(1-6), 1–7 (2000).
[CrossRef]

Lindwasser, O. W.

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

Lippincott-Schwartz, J.

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

Liu, C. K.

Lukyanchuk, B.

H. Wang, L. Shi, B. Lukyanchuk, C. Sheppard, and C. T. Chong, “Creation of a needle of longitudinally polarized light in vacuum using binary optics,” Nat. Photonics 2(8), 501–505 (2008).
[CrossRef]

Morigaki, K.

Okazaki, T.

Olenych, S.

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

Patterson, G. H.

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

Piché, M.

Quabis, S.

R. Dorn, S. Quabis, and G. Leuchs, “Sharper focus for a radially polarized light beam,” Phys. Rev. Lett. 91(23), 233901 (2003).
[CrossRef] [PubMed]

S. Quabis, R. Dorn, M. Eberler, O. Glöckl, and G. Leuchs, “Focusing light to a tighter spot,” Opt. Commun. 179(1-6), 1–7 (2000).
[CrossRef]

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. Lond. A Math. Phys. Sci. 253(1274), 358–379 (1959).
[CrossRef]

Rust, M. J.

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

Ryosuke, M.

Saad Hamady, S. O.

Sato, S.

Sheppard, C.

H. Wang, L. Shi, B. Lukyanchuk, C. Sheppard, and C. T. Chong, “Creation of a needle of longitudinally polarized light in vacuum using binary optics,” Nat. Photonics 2(8), 501–505 (2008).
[CrossRef]

Sheppard, C. J. R.

C. J. R. Sheppard and P. Török, “An electromagnetic theory of imaging in fluorescence microscopy, and imaging in polarization fluorescence microscopy,” Bioimaging 5(4), 205–218 (1997).
[CrossRef]

Shi, L.

H. Wang, L. Shi, B. Lukyanchuk, C. Sheppard, and C. T. Chong, “Creation of a needle of longitudinally polarized light in vacuum using binary optics,” Nat. Photonics 2(8), 501–505 (2008).
[CrossRef]

Sougrat, R.

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

Sun, C. C.

Terakado, G.

Török, P.

P. D. Higdon, P. Török, and T. Wilson, “Imaging properties of high aperture multiphoton fluorescence scanning optical microscopes,” J. Microsc. 193(2), 127–141 (1999).
[CrossRef]

P. Török, P. D. Higdon, and T. Wilson, “Theory for confocal and conventional microscopes imaging small dielectric scatterers,” J. Mod. Opt. 45(8), 1681–1698 (1998).
[CrossRef]

C. J. R. Sheppard and P. Török, “An electromagnetic theory of imaging in fluorescence microscopy, and imaging in polarization fluorescence microscopy,” Bioimaging 5(4), 205–218 (1997).
[CrossRef]

Tovar, A. A.

Wang, H.

H. Wang, L. Shi, B. Lukyanchuk, C. Sheppard, and C. T. Chong, “Creation of a needle of longitudinally polarized light in vacuum using binary optics,” Nat. Photonics 2(8), 501–505 (2008).
[CrossRef]

Watanabe, K.

Wichmann, J.

Willig, K. I.

B. Hein, K. I. Willig, and S. W. Hell, “Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell,” Proc. Natl. Acad. Sci. U.S.A. 105(38), 14271–14276 (2008).
[CrossRef] [PubMed]

Wilson, T.

P. D. Higdon, P. Török, and T. Wilson, “Imaging properties of high aperture multiphoton fluorescence scanning optical microscopes,” J. Microsc. 193(2), 127–141 (1999).
[CrossRef]

P. Török, P. D. Higdon, and T. Wilson, “Theory for confocal and conventional microscopes imaging small dielectric scatterers,” J. Mod. Opt. 45(8), 1681–1698 (1998).
[CrossRef]

T. Wilson and A. R. Carlini, “Size of the detector in confocal imaging systems,” Opt. Lett. 12(4), 227–229 (1987).
[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. Lond. A Math. Phys. Sci. 253(1274), 358–379 (1959).
[CrossRef]

Zhan, Q.

Zhuang, X.

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

Adv. Opt. Photon. (1)

Appl. Opt. (2)

Bioimaging (1)

C. J. R. Sheppard and P. Török, “An electromagnetic theory of imaging in fluorescence microscopy, and imaging in polarization fluorescence microscopy,” Bioimaging 5(4), 205–218 (1997).
[CrossRef]

J. Microsc. (2)

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

P. D. Higdon, P. Török, and T. Wilson, “Imaging properties of high aperture multiphoton fluorescence scanning optical microscopes,” J. Microsc. 193(2), 127–141 (1999).
[CrossRef]

J. Mod. Opt. (1)

P. Török, P. D. Higdon, and T. Wilson, “Theory for confocal and conventional microscopes imaging small dielectric scatterers,” J. Mod. Opt. 45(8), 1681–1698 (1998).
[CrossRef]

J. Opt. Soc. Am. A (2)

Microsc. Res. Tech. (1)

J. Kim, D. C. Kim, and S. H. Back, “Demonstration of high lateral resolution in laser confocal microscopy using annular and radially polarized light,” Microsc. Res. Tech. 72(6), 441–446 (2009).
[CrossRef] [PubMed]

Nat. Methods (1)

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

Nat. Photonics (1)

H. Wang, L. Shi, B. Lukyanchuk, C. Sheppard, and C. T. Chong, “Creation of a needle of longitudinally polarized light in vacuum using binary optics,” Nat. Photonics 2(8), 501–505 (2008).
[CrossRef]

Opt. Commun. (2)

L. E. Helseth, “Roles of polarization, phase and amplitude in solid immersion lens systems,” Opt. Commun. 191(3-6), 161–172 (2001).
[CrossRef]

S. Quabis, R. Dorn, M. Eberler, O. Glöckl, and G. Leuchs, “Focusing light to a tighter spot,” Opt. Commun. 179(1-6), 1–7 (2000).
[CrossRef]

Opt. Lett. (4)

Phys. Rev. Lett. (1)

R. Dorn, S. Quabis, and G. Leuchs, “Sharper focus for a radially polarized light beam,” Phys. Rev. Lett. 91(23), 233901 (2003).
[CrossRef] [PubMed]

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

B. Hein, K. I. Willig, and S. W. Hell, “Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell,” Proc. Natl. Acad. Sci. U.S.A. 105(38), 14271–14276 (2008).
[CrossRef] [PubMed]

Proc. R. Soc. Lond. A Math. Phys. Sci. (1)

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems II. Structure of the image field in an aplanatic system,” Proc. R. Soc. Lond. A Math. Phys. Sci. 253(1274), 358–379 (1959).
[CrossRef]

Science (1)

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

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Figures (5)

Fig. 1
Fig. 1

Schematic of beam conversion using LCDs.

Fig. 2
Fig. 2

(a) and (b) show the calculated intensity distributions in the focal plane (z = 0) for focusing of the LP (polarized along the x-axis) and converted HRP beams, respectively. In these calculations, NA = 1.2 and the refractive index of the surroundings (n = 1.33) were used. (c) The corresponding intensity profiles along the radial direction (x- and y-axes). The dashed and dashed-dotted curves indicate the intensity profiles of a focal spot of an LP beam in (a) along the x- and y-axes, respectively. The red solid curve shows the intensity profile of the HRP beam focusing. The inset in (c) shows the magnified profiles near the focus. (d) and (e) show the calculated intensity distributions in the z-x plane for the focusing of the LP and HRP beams, respectively (f) The corresponding intensity profiles along the z-axis in (d) and (e). The dashed and solid curves in (f) are the intensity profiles for LP and HRP beams, respectively. The scale bars in (a), (b), (d), and (e) are 1λ ( = λ0/n, where λ0 is the wavelength of a focused light in a vacuum).

Fig. 3
Fig. 3

Images and intensity profiles of a 0.17 μm fluorescent bead for the LP [(a) and (b)] and HRP [(c) and (d)] beams with CA sizes of 800 and 100 μm. Measured intensity profiles along the horizontal axis were plotted as gray dotted curves. Red curves indicate the numerically predicted profiles. The insets show the representative images of the bead for each condition. (e) The variation of the intensity profile for HRP beam excitation with different CA sizes.

Fig. 4
Fig. 4

Confocal images of aggregated fluorescent 0.17 μm beads by LP [(a) and (b)] and HRP beams [(c) and (d)]. (a) and (c) are images in the focal plane. (b) and (d) are images in the y-z plane at the position indicated by the green arrow head. The scale bar (500 nm) in (a) is valid for all of the figures.

Fig. 5
Fig. 5

Images of microtubules in a COS-7 cell with LP [(a), (b) and (d)] and HRP [(c) and (e)] beams. (b) and (c) show magnified images obtained using the LP and HRP beams, respectively, at the position indicated by the rectangle in (a). The scale bar (2 μm) in (b) is valid for (b) and (c). (d) and (e) show the magnified images at the positions indicated the rectangles in (b) and (c), respectively. The scale bar (1 μm) in (d) is valid for (d) and (e). (f) The intensity profiles along the short green line in (d) and (e) are indicated by the blue and red lines, respectively.

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