Abstract

Laser scanning microscopy allows for three-dimensional imaging of cells with molecular specific labeling. However the spatial resolution of optical microscopy is fundamentally limited by the diffraction of light. In the last two decades many techniques have been introduced to enhance the resolution of laser scanning microscopes. However most of these techniques impose strong constraints on the specimen or rely on complex optical systems. These constraints limit the applicability of resolution improvement to various imaging modalities and sample types. To overcome these limitations, we introduce here a novel approach, which we called Switching LAser Mode (SLAM) microscopy, to enhance resolution and contrast in laser scanning microscopy. SLAM microscopy relies on subtracting images obtained with dark and bright modes, and exploits the smaller dimensions of the dark spot of the azimuthally polarized TE01 mode. With this approach, resolution is improved by a factor of two in confocal microscopy. The technique is not based on complex nonlinear processes and thus requires laser power similar to that used in conventional imaging, minimizing photo-damage. The flexibility of the approach enables retrofitting in commercial confocal and two-photon microscopes and opens avenues for resolution enhancement in fluorescence-independent microscopy.

© 2013 OSA

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2012 (2)

R. Fiolka, L. Shao, E. H. Rego, M. W. Davidson, and M. G. Gustafsson, “Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination,” Proc. Natl. Acad. Sci. U.S.A.109(14), 5311–5315 (2012).
[CrossRef] [PubMed]

S. N. Khonina and I. Golub, “How low can STED go? Comparison of different write-erase beam combinations for stimulated emission depletion microscopy,” J. Opt. Soc. Am. A29(10), 2242–2246 (2012).
[CrossRef] [PubMed]

2011 (4)

Y. Kozawa, T. Hibi, A. Sato, H. Horanai, M. Kurihara, N. Hashimoto, H. Yokoyama, T. Nemoto, and S. Sato, “Lateral resolution enhancement of laser scanning microscopy by a higher-order radially polarized mode beam,” Opt. Express19(17), 15947–15954 (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. Methods8(7), 571–573 (2011).
[CrossRef] [PubMed]

S. A. Jones, S. H. Shim, J. He, and X. Zhuang, “Fast, three-dimensional super-resolution imaging of live cells,” Nat. Methods8(6), 499–505 (2011).
[CrossRef] [PubMed]

L. Shao, P. Kner, E. H. Rego, and M. G. Gustafsson, “Super-resolution 3D microscopy of live whole cells using structured illumination,” Nat. Methods8(12), 1044–1046 (2011).
[CrossRef] [PubMed]

2010 (2)

R. Kasper, B. Harke, C. Forthmann, P. Tinnefeld, S. W. Hell, and M. Sauer, “Single-molecule STED microscopy with photostable organic fluorophores,” Small6(13), 1379–1384 (2010).
[CrossRef] [PubMed]

B. R. Boruah, “Lateral resolution enhancement in confocal microscopy by vectorial aperture engineering,” Appl. Opt.49(4), 701–707 (2010).
[CrossRef] [PubMed]

2009 (6)

S. Kredel, F. Oswald, K. Nienhaus, K. Deuschle, C. Röcker, M. Wolff, R. Heilker, G. U. Nienhaus, and J. Wiedenmann, “mRuby, a bright monomeric red fluorescent protein for labeling of subcellular structures,” PLoS ONE4(2), e4391 (2009).
[CrossRef] [PubMed]

C. Labrakakis, L. E. Lorenzo, C. Bories, A. Ribeiro-da-Silva, and Y. De Koninck, “Inhibitory coupling between inhibitory interneurons in the spinal cord dorsal horn,” Mol. Pain5(1), 24 (2009).
[CrossRef] [PubMed]

E. Bélanger, S. Bégin, S. Laffray, Y. De Koninck, R. Vallée, and D. Côté, “Quantitative myelin imaging with coherent anti-Stokes Raman scattering microscopy: alleviating the excitation polarization dependence with circularly polarized laser beams,” Opt. Express17(21), 18419–18432 (2009).
[CrossRef] [PubMed]

H. Dehez, M. Piché, and Y. De Koninck, “Enhanced resolution in two-photon imaging using a TM01 laser beam at a dielectric interface,” Opt. Lett.34(23), 3601–3603 (2009).
[CrossRef] [PubMed]

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]

O. Haeberlé and B. Simon, “Saturated structured confocal microscopy with theoretically unlimited resolution,” Opt. Commun.282(18), 3657–3664 (2009).
[CrossRef]

2008 (2)

H. Shroff, C. G. Galbraith, J. A. Galbraith, and E. Betzig, “Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics,” Nat. Methods5(5), 417–423 (2008).
[CrossRef] [PubMed]

J. Stadler, C. Stanciu, C. Stupperich, and A. J. Meixner, “Tighter focusing with a parabolic mirror,” Opt. Lett.33(7), 681–683 (2008).
[CrossRef] [PubMed]

2006 (5)

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,” Science313(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. Methods3(10), 793–796 (2006).
[CrossRef] [PubMed]

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

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods3(1), 47–53 (2006).
[CrossRef] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A.103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

2005 (2)

A. Hudmon, E. Lebel, H. Roy, A. Sik, H. Schulman, M. N. Waxham, and P. De Koninck, “A mechanism for Ca2+/calmodulin-dependent protein kinase II clustering at synaptic and nonsynaptic sites based on self-association,” J. Neurosci.25(30), 6971–6983 (2005).
[CrossRef] [PubMed]

H. Balci, T. Ha, H. L. Sweeney, and P. R. Selvin, “Interhead distance measurements in myosin VI via SHRImP support a simplified hand-over-hand model,” Biophys. J.89(1), 413–417 (2005).
[CrossRef] [PubMed]

2003 (3)

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, “Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization,” Science300(5628), 2061–2065 (2003).
[CrossRef] [PubMed]

T. A. Pologruto, B. L. Sabatini, and K. Svoboda, “ScanImage: flexible software for operating laser scanning microscopes,” Biomed. Eng. Online2(1), 13 (2003).
[CrossRef] [PubMed]

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

Andrei, M. A.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A.103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

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]

Balci, H.

H. Balci, T. Ha, H. L. Sweeney, and P. R. Selvin, “Interhead distance measurements in myosin VI via SHRImP support a simplified hand-over-hand model,” Biophys. J.89(1), 413–417 (2005).
[CrossRef] [PubMed]

Bates, M.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods3(10), 793–796 (2006).
[CrossRef] [PubMed]

Beaurepaire, E.

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods3(1), 47–53 (2006).
[CrossRef] [PubMed]

Bégin, S.

Bélanger, E.

Betzig, E.

H. Shroff, C. G. Galbraith, J. A. Galbraith, and E. Betzig, “Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics,” Nat. Methods5(5), 417–423 (2008).
[CrossRef] [PubMed]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(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,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Bories, C.

C. Labrakakis, L. E. Lorenzo, C. Bories, A. Ribeiro-da-Silva, and Y. De Koninck, “Inhibitory coupling between inhibitory interneurons in the spinal cord dorsal horn,” Mol. Pain5(1), 24 (2009).
[CrossRef] [PubMed]

Boruah, B. R.

Combettes, L.

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods3(1), 47–53 (2006).
[CrossRef] [PubMed]

Côté, D.

Davidson, M. W.

R. Fiolka, L. Shao, E. H. Rego, M. W. Davidson, and M. G. Gustafsson, “Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination,” Proc. Natl. Acad. Sci. U.S.A.109(14), 5311–5315 (2012).
[CrossRef] [PubMed]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

De Koninck, P.

A. Hudmon, E. Lebel, H. Roy, A. Sik, H. Schulman, M. N. Waxham, and P. De Koninck, “A mechanism for Ca2+/calmodulin-dependent protein kinase II clustering at synaptic and nonsynaptic sites based on self-association,” J. Neurosci.25(30), 6971–6983 (2005).
[CrossRef] [PubMed]

De Koninck, Y.

Débarre, D.

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods3(1), 47–53 (2006).
[CrossRef] [PubMed]

Dehez, H.

Deuschle, K.

S. Kredel, F. Oswald, K. Nienhaus, K. Deuschle, C. Röcker, M. Wolff, R. Heilker, G. U. Nienhaus, and J. Wiedenmann, “mRuby, a bright monomeric red fluorescent protein for labeling of subcellular structures,” PLoS ONE4(2), e4391 (2009).
[CrossRef] [PubMed]

Donnert, G.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A.103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

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]

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. Methods8(7), 571–573 (2011).
[CrossRef] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A.103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

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. Methods8(7), 571–573 (2011).
[CrossRef] [PubMed]

Fabre, A.

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods3(1), 47–53 (2006).
[CrossRef] [PubMed]

Fiolka, R.

R. Fiolka, L. Shao, E. H. Rego, M. W. Davidson, and M. G. Gustafsson, “Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination,” Proc. Natl. Acad. Sci. U.S.A.109(14), 5311–5315 (2012).
[CrossRef] [PubMed]

Forkey, J. N.

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, “Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization,” Science300(5628), 2061–2065 (2003).
[CrossRef] [PubMed]

Forthmann, C.

R. Kasper, B. Harke, C. Forthmann, P. Tinnefeld, S. W. Hell, and M. Sauer, “Single-molecule STED microscopy with photostable organic fluorophores,” Small6(13), 1379–1384 (2010).
[CrossRef] [PubMed]

Galbraith, C. G.

H. Shroff, C. G. Galbraith, J. A. Galbraith, and E. Betzig, “Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics,” Nat. Methods5(5), 417–423 (2008).
[CrossRef] [PubMed]

Galbraith, J. A.

H. Shroff, C. G. Galbraith, J. A. Galbraith, and E. Betzig, “Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics,” Nat. Methods5(5), 417–423 (2008).
[CrossRef] [PubMed]

Girirajan, T. P.

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

Goldman, Y. E.

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, “Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization,” Science300(5628), 2061–2065 (2003).
[CrossRef] [PubMed]

Golub, I.

Gustafsson, M. G.

R. Fiolka, L. Shao, E. H. Rego, M. W. Davidson, and M. G. Gustafsson, “Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination,” Proc. Natl. Acad. Sci. U.S.A.109(14), 5311–5315 (2012).
[CrossRef] [PubMed]

L. Shao, P. Kner, E. H. Rego, and M. G. Gustafsson, “Super-resolution 3D microscopy of live whole cells using structured illumination,” Nat. Methods8(12), 1044–1046 (2011).
[CrossRef] [PubMed]

Ha, T.

H. Balci, T. Ha, H. L. Sweeney, and P. R. Selvin, “Interhead distance measurements in myosin VI via SHRImP support a simplified hand-over-hand model,” Biophys. J.89(1), 413–417 (2005).
[CrossRef] [PubMed]

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, “Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization,” Science300(5628), 2061–2065 (2003).
[CrossRef] [PubMed]

Haeberlé, O.

O. Haeberlé and B. Simon, “Saturated structured confocal microscopy with theoretically unlimited resolution,” Opt. Commun.282(18), 3657–3664 (2009).
[CrossRef]

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. Methods8(7), 571–573 (2011).
[CrossRef] [PubMed]

Harke, B.

R. Kasper, B. Harke, C. Forthmann, P. Tinnefeld, S. W. Hell, and M. Sauer, “Single-molecule STED microscopy with photostable organic fluorophores,” Small6(13), 1379–1384 (2010).
[CrossRef] [PubMed]

Hashimoto, N.

He, J.

S. A. Jones, S. H. Shim, J. He, and X. Zhuang, “Fast, three-dimensional super-resolution imaging of live cells,” Nat. Methods8(6), 499–505 (2011).
[CrossRef] [PubMed]

Heilker, R.

S. Kredel, F. Oswald, K. Nienhaus, K. Deuschle, C. Röcker, M. Wolff, R. Heilker, G. U. Nienhaus, and J. Wiedenmann, “mRuby, a bright monomeric red fluorescent protein for labeling of subcellular structures,” PLoS ONE4(2), e4391 (2009).
[CrossRef] [PubMed]

Hell, S. W.

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. Methods8(7), 571–573 (2011).
[CrossRef] [PubMed]

R. Kasper, B. Harke, C. Forthmann, P. Tinnefeld, S. W. Hell, and M. Sauer, “Single-molecule STED microscopy with photostable organic fluorophores,” Small6(13), 1379–1384 (2010).
[CrossRef] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A.103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

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,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Hess, S. T.

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

Hibi, T.

Horanai, H.

Hudmon, A.

A. Hudmon, E. Lebel, H. Roy, A. Sik, H. Schulman, M. N. Waxham, and P. De Koninck, “A mechanism for Ca2+/calmodulin-dependent protein kinase II clustering at synaptic and nonsynaptic sites based on self-association,” J. Neurosci.25(30), 6971–6983 (2005).
[CrossRef] [PubMed]

Jahn, R.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A.103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Jones, S. A.

S. A. Jones, S. H. Shim, J. He, and X. Zhuang, “Fast, three-dimensional super-resolution imaging of live cells,” Nat. Methods8(6), 499–505 (2011).
[CrossRef] [PubMed]

Kasper, R.

R. Kasper, B. Harke, C. Forthmann, P. Tinnefeld, S. W. Hell, and M. Sauer, “Single-molecule STED microscopy with photostable organic fluorophores,” Small6(13), 1379–1384 (2010).
[CrossRef] [PubMed]

Keller, J.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A.103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Khonina, S. N.

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]

Kner, P.

L. Shao, P. Kner, E. H. Rego, and M. G. Gustafsson, “Super-resolution 3D microscopy of live whole cells using structured illumination,” Nat. Methods8(12), 1044–1046 (2011).
[CrossRef] [PubMed]

Kozawa, Y.

Kredel, S.

S. Kredel, F. Oswald, K. Nienhaus, K. Deuschle, C. Röcker, M. Wolff, R. Heilker, G. U. Nienhaus, and J. Wiedenmann, “mRuby, a bright monomeric red fluorescent protein for labeling of subcellular structures,” PLoS ONE4(2), e4391 (2009).
[CrossRef] [PubMed]

Kurihara, M.

Labrakakis, C.

C. Labrakakis, L. E. Lorenzo, C. Bories, A. Ribeiro-da-Silva, and Y. De Koninck, “Inhibitory coupling between inhibitory interneurons in the spinal cord dorsal horn,” Mol. Pain5(1), 24 (2009).
[CrossRef] [PubMed]

Laffray, S.

Lebel, E.

A. Hudmon, E. Lebel, H. Roy, A. Sik, H. Schulman, M. N. Waxham, and P. De Koninck, “A mechanism for Ca2+/calmodulin-dependent protein kinase II clustering at synaptic and nonsynaptic sites based on self-association,” J. Neurosci.25(30), 6971–6983 (2005).
[CrossRef] [PubMed]

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]

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,” Science313(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,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Lorenzo, L. E.

C. Labrakakis, L. E. Lorenzo, C. Bories, A. Ribeiro-da-Silva, and Y. De Koninck, “Inhibitory coupling between inhibitory interneurons in the spinal cord dorsal horn,” Mol. Pain5(1), 24 (2009).
[CrossRef] [PubMed]

Lührmann, R.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A.103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Mason, M. D.

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

McKinney, S. A.

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, “Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization,” Science300(5628), 2061–2065 (2003).
[CrossRef] [PubMed]

Medda, R.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A.103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Meixner, A. J.

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. Methods8(7), 571–573 (2011).
[CrossRef] [PubMed]

Nemoto, T.

Nienhaus, G. U.

S. Kredel, F. Oswald, K. Nienhaus, K. Deuschle, C. Röcker, M. Wolff, R. Heilker, G. U. Nienhaus, and J. Wiedenmann, “mRuby, a bright monomeric red fluorescent protein for labeling of subcellular structures,” PLoS ONE4(2), e4391 (2009).
[CrossRef] [PubMed]

Nienhaus, K.

S. Kredel, F. Oswald, K. Nienhaus, K. Deuschle, C. Röcker, M. Wolff, R. Heilker, G. U. Nienhaus, and J. Wiedenmann, “mRuby, a bright monomeric red fluorescent protein for labeling of subcellular structures,” PLoS ONE4(2), e4391 (2009).
[CrossRef] [PubMed]

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,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Oswald, F.

S. Kredel, F. Oswald, K. Nienhaus, K. Deuschle, C. Röcker, M. Wolff, R. Heilker, G. U. Nienhaus, and J. Wiedenmann, “mRuby, a bright monomeric red fluorescent protein for labeling of subcellular structures,” PLoS ONE4(2), e4391 (2009).
[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,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Pena, A. M.

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods3(1), 47–53 (2006).
[CrossRef] [PubMed]

Piché, M.

Pologruto, T. A.

T. A. Pologruto, B. L. Sabatini, and K. Svoboda, “ScanImage: flexible software for operating laser scanning microscopes,” Biomed. Eng. Online2(1), 13 (2003).
[CrossRef] [PubMed]

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]

Rego, E. H.

R. Fiolka, L. Shao, E. H. Rego, M. W. Davidson, and M. G. Gustafsson, “Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination,” Proc. Natl. Acad. Sci. U.S.A.109(14), 5311–5315 (2012).
[CrossRef] [PubMed]

L. Shao, P. Kner, E. H. Rego, and M. G. Gustafsson, “Super-resolution 3D microscopy of live whole cells using structured illumination,” Nat. Methods8(12), 1044–1046 (2011).
[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. Methods8(7), 571–573 (2011).
[CrossRef] [PubMed]

Ribeiro-da-Silva, A.

C. Labrakakis, L. E. Lorenzo, C. Bories, A. Ribeiro-da-Silva, and Y. De Koninck, “Inhibitory coupling between inhibitory interneurons in the spinal cord dorsal horn,” Mol. Pain5(1), 24 (2009).
[CrossRef] [PubMed]

Rizzoli, S. O.

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A.103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Röcker, C.

S. Kredel, F. Oswald, K. Nienhaus, K. Deuschle, C. Röcker, M. Wolff, R. Heilker, G. U. Nienhaus, and J. Wiedenmann, “mRuby, a bright monomeric red fluorescent protein for labeling of subcellular structures,” PLoS ONE4(2), e4391 (2009).
[CrossRef] [PubMed]

Roy, H.

A. Hudmon, E. Lebel, H. Roy, A. Sik, H. Schulman, M. N. Waxham, and P. De Koninck, “A mechanism for Ca2+/calmodulin-dependent protein kinase II clustering at synaptic and nonsynaptic sites based on self-association,” J. Neurosci.25(30), 6971–6983 (2005).
[CrossRef] [PubMed]

Rust, M. J.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods3(10), 793–796 (2006).
[CrossRef] [PubMed]

Sabatini, B. L.

T. A. Pologruto, B. L. Sabatini, and K. Svoboda, “ScanImage: flexible software for operating laser scanning microscopes,” Biomed. Eng. Online2(1), 13 (2003).
[CrossRef] [PubMed]

Sato, A.

Sato, S.

Sauer, M.

R. Kasper, B. Harke, C. Forthmann, P. Tinnefeld, S. W. Hell, and M. Sauer, “Single-molecule STED microscopy with photostable organic fluorophores,” Small6(13), 1379–1384 (2010).
[CrossRef] [PubMed]

Schanne-Klein, M. C.

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods3(1), 47–53 (2006).
[CrossRef] [PubMed]

Schulman, H.

A. Hudmon, E. Lebel, H. Roy, A. Sik, H. Schulman, M. N. Waxham, and P. De Koninck, “A mechanism for Ca2+/calmodulin-dependent protein kinase II clustering at synaptic and nonsynaptic sites based on self-association,” J. Neurosci.25(30), 6971–6983 (2005).
[CrossRef] [PubMed]

Selvin, P. R.

H. Balci, T. Ha, H. L. Sweeney, and P. R. Selvin, “Interhead distance measurements in myosin VI via SHRImP support a simplified hand-over-hand model,” Biophys. J.89(1), 413–417 (2005).
[CrossRef] [PubMed]

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, “Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization,” Science300(5628), 2061–2065 (2003).
[CrossRef] [PubMed]

Shao, L.

R. Fiolka, L. Shao, E. H. Rego, M. W. Davidson, and M. G. Gustafsson, “Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination,” Proc. Natl. Acad. Sci. U.S.A.109(14), 5311–5315 (2012).
[CrossRef] [PubMed]

L. Shao, P. Kner, E. H. Rego, and M. G. Gustafsson, “Super-resolution 3D microscopy of live whole cells using structured illumination,” Nat. Methods8(12), 1044–1046 (2011).
[CrossRef] [PubMed]

Shim, S. H.

S. A. Jones, S. H. Shim, J. He, and X. Zhuang, “Fast, three-dimensional super-resolution imaging of live cells,” Nat. Methods8(6), 499–505 (2011).
[CrossRef] [PubMed]

Shroff, H.

H. Shroff, C. G. Galbraith, J. A. Galbraith, and E. Betzig, “Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics,” Nat. Methods5(5), 417–423 (2008).
[CrossRef] [PubMed]

Sik, A.

A. Hudmon, E. Lebel, H. Roy, A. Sik, H. Schulman, M. N. Waxham, and P. De Koninck, “A mechanism for Ca2+/calmodulin-dependent protein kinase II clustering at synaptic and nonsynaptic sites based on self-association,” J. Neurosci.25(30), 6971–6983 (2005).
[CrossRef] [PubMed]

Simon, B.

O. Haeberlé and B. Simon, “Saturated structured confocal microscopy with theoretically unlimited resolution,” Opt. Commun.282(18), 3657–3664 (2009).
[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,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Stadler, J.

Stanciu, C.

Stupperich, C.

Supatto, W.

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods3(1), 47–53 (2006).
[CrossRef] [PubMed]

Svoboda, K.

T. A. Pologruto, B. L. Sabatini, and K. Svoboda, “ScanImage: flexible software for operating laser scanning microscopes,” Biomed. Eng. Online2(1), 13 (2003).
[CrossRef] [PubMed]

Sweeney, H. L.

H. Balci, T. Ha, H. L. Sweeney, and P. R. Selvin, “Interhead distance measurements in myosin VI via SHRImP support a simplified hand-over-hand model,” Biophys. J.89(1), 413–417 (2005).
[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. Methods8(7), 571–573 (2011).
[CrossRef] [PubMed]

Tinnefeld, P.

R. Kasper, B. Harke, C. Forthmann, P. Tinnefeld, S. W. Hell, and M. Sauer, “Single-molecule STED microscopy with photostable organic fluorophores,” Small6(13), 1379–1384 (2010).
[CrossRef] [PubMed]

Tordjmann, T.

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods3(1), 47–53 (2006).
[CrossRef] [PubMed]

Vallée, R.

Vicidomini, 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. Methods8(7), 571–573 (2011).
[CrossRef] [PubMed]

Waxham, M. N.

A. Hudmon, E. Lebel, H. Roy, A. Sik, H. Schulman, M. N. Waxham, and P. De Koninck, “A mechanism for Ca2+/calmodulin-dependent protein kinase II clustering at synaptic and nonsynaptic sites based on self-association,” J. Neurosci.25(30), 6971–6983 (2005).
[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. Methods8(7), 571–573 (2011).
[CrossRef] [PubMed]

Wiedenmann, J.

S. Kredel, F. Oswald, K. Nienhaus, K. Deuschle, C. Röcker, M. Wolff, R. Heilker, G. U. Nienhaus, and J. Wiedenmann, “mRuby, a bright monomeric red fluorescent protein for labeling of subcellular structures,” PLoS ONE4(2), e4391 (2009).
[CrossRef] [PubMed]

Wolff, M.

S. Kredel, F. Oswald, K. Nienhaus, K. Deuschle, C. Röcker, M. Wolff, R. Heilker, G. U. Nienhaus, and J. Wiedenmann, “mRuby, a bright monomeric red fluorescent protein for labeling of subcellular structures,” PLoS ONE4(2), e4391 (2009).
[CrossRef] [PubMed]

Yildiz, A.

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, “Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization,” Science300(5628), 2061–2065 (2003).
[CrossRef] [PubMed]

Yokoyama, H.

Zhuang, X.

S. A. Jones, S. H. Shim, J. He, and X. Zhuang, “Fast, three-dimensional super-resolution imaging of live cells,” Nat. Methods8(6), 499–505 (2011).
[CrossRef] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods3(10), 793–796 (2006).
[CrossRef] [PubMed]

Appl. Opt. (1)

Biomed. Eng. Online (1)

T. A. Pologruto, B. L. Sabatini, and K. Svoboda, “ScanImage: flexible software for operating laser scanning microscopes,” Biomed. Eng. Online2(1), 13 (2003).
[CrossRef] [PubMed]

Biophys. J. (2)

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

H. Balci, T. Ha, H. L. Sweeney, and P. R. Selvin, “Interhead distance measurements in myosin VI via SHRImP support a simplified hand-over-hand model,” Biophys. J.89(1), 413–417 (2005).
[CrossRef] [PubMed]

J. Neurosci. (1)

A. Hudmon, E. Lebel, H. Roy, A. Sik, H. Schulman, M. N. Waxham, and P. De Koninck, “A mechanism for Ca2+/calmodulin-dependent protein kinase II clustering at synaptic and nonsynaptic sites based on self-association,” J. Neurosci.25(30), 6971–6983 (2005).
[CrossRef] [PubMed]

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

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]

Mol. Pain (1)

C. Labrakakis, L. E. Lorenzo, C. Bories, A. Ribeiro-da-Silva, and Y. De Koninck, “Inhibitory coupling between inhibitory interneurons in the spinal cord dorsal horn,” Mol. Pain5(1), 24 (2009).
[CrossRef] [PubMed]

Nat. Methods (6)

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods3(10), 793–796 (2006).
[CrossRef] [PubMed]

D. Débarre, W. Supatto, A. M. Pena, A. Fabre, T. Tordjmann, L. Combettes, M. C. Schanne-Klein, and E. Beaurepaire, “Imaging lipid bodies in cells and tissues using third-harmonic generation microscopy,” Nat. Methods3(1), 47–53 (2006).
[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. Methods8(7), 571–573 (2011).
[CrossRef] [PubMed]

H. Shroff, C. G. Galbraith, J. A. Galbraith, and E. Betzig, “Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics,” Nat. Methods5(5), 417–423 (2008).
[CrossRef] [PubMed]

S. A. Jones, S. H. Shim, J. He, and X. Zhuang, “Fast, three-dimensional super-resolution imaging of live cells,” Nat. Methods8(6), 499–505 (2011).
[CrossRef] [PubMed]

L. Shao, P. Kner, E. H. Rego, and M. G. Gustafsson, “Super-resolution 3D microscopy of live whole cells using structured illumination,” Nat. Methods8(12), 1044–1046 (2011).
[CrossRef] [PubMed]

Opt. Commun. (1)

O. Haeberlé and B. Simon, “Saturated structured confocal microscopy with theoretically unlimited resolution,” Opt. Commun.282(18), 3657–3664 (2009).
[CrossRef]

Opt. Express (2)

Opt. Lett. (2)

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]

PLoS ONE (1)

S. Kredel, F. Oswald, K. Nienhaus, K. Deuschle, C. Röcker, M. Wolff, R. Heilker, G. U. Nienhaus, and J. Wiedenmann, “mRuby, a bright monomeric red fluorescent protein for labeling of subcellular structures,” PLoS ONE4(2), e4391 (2009).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. U.S.A. (2)

R. Fiolka, L. Shao, E. H. Rego, M. W. Davidson, and M. G. Gustafsson, “Time-lapse two-color 3D imaging of live cells with doubled resolution using structured illumination,” Proc. Natl. Acad. Sci. U.S.A.109(14), 5311–5315 (2012).
[CrossRef] [PubMed]

G. Donnert, J. Keller, R. Medda, M. A. Andrei, S. O. Rizzoli, R. Lührmann, R. Jahn, C. Eggeling, and S. W. Hell, “Macromolecular-scale resolution in biological fluorescence microscopy,” Proc. Natl. Acad. Sci. U.S.A.103(31), 11440–11445 (2006).
[CrossRef] [PubMed]

Science (2)

A. Yildiz, J. N. Forkey, S. A. McKinney, T. Ha, Y. E. Goldman, and P. R. Selvin, “Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization,” Science300(5628), 2061–2065 (2003).
[CrossRef] [PubMed]

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Small (1)

R. Kasper, B. Harke, C. Forthmann, P. Tinnefeld, S. W. Hell, and M. Sauer, “Single-molecule STED microscopy with photostable organic fluorophores,” Small6(13), 1379–1384 (2010).
[CrossRef] [PubMed]

Other (3)

B. Richards and E. Wolf, “Electromagnetic diffraction in optical systems. 2. Structure of the image field in an aplanatic system,” Proc. R. Soc. A 253, 358–379 (1959).

L. Novotny and B. Hecht, Principles of Nano-Optics, (Cambridge University, 2007).

A. Diaspro, Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances, (Wiley-Liss, 2002).

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Figures (8)

Fig. 1
Fig. 1

Geometry of the aplanetic lens (microscope objective) and definitions of the coordinates used in the theoretical analysis.

Fig. 2
Fig. 2

Theoretical calculation of the point spread function obtained in SLAM microscopy with a 1.4 NA oil immersion objective in the mounting medium. (a) PSF obtained with a circularly polarized Gaussian beam. (b) PSF obtained with the azimuthally polarized TE01 laser beam. (c) PSF of SLAM microscopy using theoretical PSFs given in (a) and (b) and a g factor of 0.65. (d,e) Intensity profiles along the horizontal direction of the PSFs obtained with the Gaussian beam (black), the TE01 laser beam (blue) and SLAM (green). In (e), the negative values introduced by the subtraction process are set to zero because they do not have a physical meaning and are created outside of the object. Scale bar: λ.

Fig. 3
Fig. 3

Theoretical calculation of resolution enhancement using SLAM microscopy. Theoretical images of four point sources using (a,b,c,e) conventional confocal imaging and (d) confocal SLAM microscopy. (a) Image obtained with a linearly polarized Gaussian beam of point sources separated by the limit of resolution along the direction of polarization. (b) Same as (a), for the resolution limit along the perpendicular direction. (c) Same as (a) and (b), but for a circularly polarized Gaussian beam. (d) Image obtained at the resolution limit of SLAM microscopy; (e) image obtained of the same four point sources as in (d) using conventional confocal microscopy. Scale bar 500 nm. (a-d) The resolution limits were defined by a 75% contrast between the peaks and the valley between the peaks.

Fig. 4
Fig. 4

Experimental setup for SLAM microscopy. Two components are placed between the laser source and the microscope: a mode converter to convert the Gaussian laser beam into an azimuthally polarized laser beam, and an optical switch based on rotating mirrors.

Fig. 5
Fig. 5

Experimental demonstration of resolution enhancement using SLAM microscopy: comparison between images of sparsely dispersed 100-nm diameter gold nano-particles imaged by (a) scanning electron microscopy, (b) conventional reflectance microscopy (“OFF”) and (c) SLAM microscopy (“ON”). (d) Intensity profiles evaluated along two close nano-particless in SEM (red), reflectance microscopy (dark) and SLAM reflectance microscopy (green). The orange lines indicate the centers of the nano-particless in the SEM image. Scale bars: 500 nm.

Fig. 6
Fig. 6

Experimental characterization of resolution enhancement using confocal SLAM microscopy: images of 100-nm diameter fluorescent beads. (a) Experimental (dotted lines) and theoretical (solid lines) images of bright beam (dark), dark beam (blue) and SLAM (green). The intensity profiles were evaluated along the direction perpendicular to the polarization. (b,c) Demonstration of resolution enhancement with a higher concentration of beads: comparison between conventional confocal microscope (“OFF”) and confocal SLAM images (“ON”) Scale bars: 500 nm.

Fig. 7
Fig. 7

Resolution and contrast enhancement in cell cultures: comparison between conventional confocal microscope (“OFF”) and confocal SLAM images (“ON”). (a,b) Tubulin labeling of neurons by immunohistochemistry (Alexa 546); detection of new features (close fibers) using SLAM microsopy. (c,d) m-Ruby transfected neurons; improved dendritic spine morphology analysis with SLAM microscopy. Image scale bars: 1 μm. Profile scale bars: 500 nm.

Fig. 8
Fig. 8

Two-photon SLAM imaging. (a) Experimental (dotted lines) and theoretical (solid lines) intensity profiles of bright beam (dark), dark beam (blue) and SLAM (green) evaluated along the direction perpendicular to the polarization. (b,c) Demonstration of resolution enhancement with clusters of fluorescent beads: comparison between conventional two-photon excitation microscopy (“OFF”) and two-photon SLAM images (“ON”). (d) Two-photon imaging of neuronal dendritic spines in 250-μm thick brain slices. (e) Hippocampal pyramidal neurons were labeled by micro-injection of Lucifer-Yellow. (a,b) Scale bar: 500 nm. (d) Image scale bars: 1 μm. Profile scale bars: 500 nm.

Tables (1)

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Table 1 Comparison between theoretical dark beam and bright beam diameters

Equations (25)

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E(r,ϕ,z)= E o 2π Ω q(α)A(α,β)exp( jkr )dΩ ,
0 (α,β)=exp( (fsinα) 2 / w 2 ),
a ^ (α,β)=[ a ^ x a ^ r ] a ^ α +[ a ^ x a ^ β ] a ^ β = a ^ α (α,β)cosβ a ^ β (β)sinβ,
a ^ (α,β)= a ^ x 1 2 [ (cosα+1)+(cosα1)cos2β ] + a ^ y 1 2 (cosα1)sin2β a ^ z sinαcosβ,
a ^ α (α,β)= a ^ x cosαcosβ+ a ^ y cosαsinβ a ^ z sinα,
a ^ β (β)= a ^ x sinβ+ a ^ y cosβ,
E(r,ϕ,z)= E o 2π n 0 2π 0 α max cos α 1/2 exp ( (fsinα) 2 / w 2 ) sin α ×( a ^ α cosβ a ^ β sinβ )exp[ jk( zcosα+rsinαcos(ϕβ) ) ]d α dβ,
0 2π exp[ jγcos(ϕβ) ]{ cos(mβ) sin(mβ) }dβ =2π j m J m (γ){ cos(mϕ) sin(mϕ) },
E x (r,ϕ,z)= E o 2 n 0 α max cos α 1/2 sinαexp( (fsinα) 2 / w 2 )exp(jkzcosα) ×[ (1+cosα) J 0 (krsinα)+(1cosα) J 2 (krsinα)cos2ϕ ]dα,
E y (r,ϕ,z)= E o 2 n 0 α max cos α 1/2 sinαexp( (fsinα) 2 / w 2 )exp(jkzcosα) ×(1cosα) J 2 (krsinα)sin2 ϕ dα,
E z (r,ϕ,z)=j E o n 0 α max cos α 1/2 sin α 2 exp( (fsinα) 2 / w 2 )exp(jkzcosα) × J 1 (krsinα)cosϕdα.
E x coverslip = E x medium ,
E y coverslip = E y medium ,
ε coverslip E z coverslip = ε medium E z medium .
E(r,ϕ,z)= E o 2π 0 2π 0 α max q(α) 0 (α)( a ^ x sinβ+ a ^ y cosβ ) ×exp[ jk( zcosα+rsinαcos(ϕβ) ) ]sin α d α dβ.
E(r,z)=j E o n f w a ^ ϕ 0 α max cos α 1/2 sin α 2 exp( (fsinα) 2 / w 2 ) × J 1 (krsinα)exp( jkzcosα )dα,
0 (α,β)= fsinα w e jβ exp( (fsinα) 2 / w 2 ).
a ^ (α,β)=[ ( a ^ x +j a ^ y ) a ^ r ] a ^ α +[ ( a ^ x +j a ^ y ) a ^ β ] a ^ β = e jβ ( a ^ α (α,β)+j a ^ β (β)),
a ^ (α,β)= a ^ x e jβ (cosαcosβjsinβ) + a ^ y e jβ (cosαsinβ+jcosβ) a ^ z e jβ sinα,
E(r,ϕ,z)= E o 2π n f w 0 2π 0 α max cos α 1/2 sin α 2 e 2jβ exp( (fsinα) 2 / w 2 ) ×( a ^ α (α,β)+j a ^ β (β))exp[ jk( zcosα+rsinαcos(ϕβ) ) ]d α dβ.
0 2π exp[ jγcos(ϕβ) ] e j2β cos β dβ =πj J 1 (γ) e jϕ πj J 3 (γ) e j3ϕ ,
0 2π exp[ jγcos(ϕβ) ] e j2β sin β dβ =π J 1 (γ) e jϕ π J 3 (γ) e j3ϕ .
E x (r,ϕ,z)=j E o 2 n f w 0 α max cos α 1/2 sin α 2 exp( (fsinα) 2 / w 2 )exp(jkzcosα) ×[ (1+cosα) J 1 (krsinα) e jϕ +(1cosα) J 3 (krsinα) e j3ϕ ]dα,
E y (r,ϕ,z)= E o 2 n f w 0 α max cos α 1/2 sin α 2 exp( (fsinα) 2 / w 2 )exp(jkzcosα) ×[ (1+cosα) J 1 (krsinα) e jϕ +(1cosα) J 3 (krsinα) e j3ϕ ]dα,
E z (r,ϕ,z)= E o n f w 0 α max cos α 1/2 exp( (fsinα) 2 / w 2 )exp(jkzcosα) ×sin α 3 J 2 (krsinα) e j2ϕ dα.

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