Abstract

Double-helix point spread function photoactivation-localization microscopy allows three-dimensional (3D) superresolution imaging of objects smaller than the optical diffraction-limit. We demonstrate polarization sensitive detection with 3D super-localization of single-molecules and unveil 3D polarization specific characteristics of single-molecules within the intracellular structure of PtK1 cells expressing photoactivatable green fluorescent protein. The system modulates orthogonal polarization components of single-molecule emissions with a single spatial light modulator and detects them separately with a single detector. Information obtained from the two polarization channels demonstrates polarization based contrast in 3D superresolution imaging. Further, we show that the 3D information from the two channels can be optimally combined to yield up to 30% improvement in localization precision relative to a single polarization channel system.

© 2009 OSA

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  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).
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2009

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[CrossRef] [PubMed]

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

S. R. P. Pavani, A. Greengard, and R. Piestun, “Three-dimensional localization with nanometer accuracy using a detector-limited double-helix point spread function system,” Appl. Phys. Lett. 95(2), 021103 (2009).
[CrossRef]

F. Aguet, S. Geissbühler, I. Märki, T. Lasser, and M. Unser, “Super-resolution orientation estimation and localization of fluorescent dipoles using 3-D steerable filters,” Opt. Express 17(8), 6829–6848 (2009).
[CrossRef] [PubMed]

2008

T. J. Gould, M. S. Gunewardene, M. V. Gudheti, V. V. Verkhusha, S.-R. Yin, J. A. Gosse, and S. T. Hess, “Nanoscale imaging of molecular positions and anisotropies,” Nat. Methods 5(12), 1027–1030 (2008).
[CrossRef] [PubMed]

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319(5864), 810–813 (2008).
[CrossRef] [PubMed]

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods 5(6), 527–529 (2008).
[CrossRef] [PubMed]

A. Vaziri, J. Tang, H. Shroff, and C. V. Shank, “Multilayer three-dimensional super resolution imaging of thick biological samples,” Proc. Natl. Acad. Sci. U.S.A. 105(51), 20221–20226 (2008).
[CrossRef] [PubMed]

M. R. Foreman, C. M. Romero, and P. Török, “Determination of the three-dimensional orientation of single molecules,” Opt. Lett. 33(9), 1020–1022 (2008).
[CrossRef] [PubMed]

S. R. P. Pavani and R. Piestun, “High-efficiency rotating point spread functions,” Opt. Express 16(5), 3484–3489 (2008).
[CrossRef] [PubMed]

S. R. P. Pavani and R. Piestun, “Three dimensional tracking of fluorescent microparticles using a photon-limited double-helix response system,” Opt. Express 16(26), 22048–22057 (2008).
[CrossRef] [PubMed]

2006

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]

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

A. Greengard, Y. Y. Schechner, and R. Piestun, “Depth from diffracted rotation,” Opt. Lett. 31(2), 181–183 (2006).
[CrossRef] [PubMed]

2004

P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, “Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions,” IEEE Trans. Nanobioscience 3(4), 237–242 (2004).
[CrossRef]

2002

G. H. Patterson and J. Lippincott-Schwartz, “A photoactivatable GFP for selective photolabeling of proteins and cells,” Science 297(5588), 1873–1877 (2002).
[CrossRef] [PubMed]

2000

B. Sick, B. Hecht, and L. Novotny, “Orientational imaging of single molecules by annular illumination,” Phys. Rev. Lett. 85(21), 4482–4485 (2000).
[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]

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]

1998

R. M. Dickson, D. J. Norris, and W. E. Moerner, “Simultaneous Imaging of Individual Molecules Aligned Both Parallel and Perpendicular to the Optic Axis,” Phys. Rev. Lett. 81(24), 5322–5325 (1998).
[CrossRef]

1996

T. Ha, T. Enderle, D. S. Chemla, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77(19), 3979–3982 (1996).
[CrossRef] [PubMed]

Aguet, F.

Bates, M.

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319(5864), 810–813 (2008).
[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]

Bennett, B. T.

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods 5(6), 527–529 (2008).
[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]

Bewersdorf, J.

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods 5(6), 527–529 (2008).
[CrossRef] [PubMed]

Biteen, J. S.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 2995–2999 (2009).
[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]

Chemla, D. S.

T. Ha, T. Enderle, D. S. Chemla, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77(19), 3979–3982 (1996).
[CrossRef] [PubMed]

Davidson, M. W.

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[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,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Dickson, R. M.

R. M. Dickson, D. J. Norris, and W. E. Moerner, “Simultaneous Imaging of Individual Molecules Aligned Both Parallel and Perpendicular to the Optic Axis,” Phys. Rev. Lett. 81(24), 5322–5325 (1998).
[CrossRef]

Dyba, M.

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

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]

Enderle, T.

T. Ha, T. Enderle, D. S. Chemla, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77(19), 3979–3982 (1996).
[CrossRef] [PubMed]

Fetter, R. D.

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[CrossRef] [PubMed]

Foreman, M. R.

Galbraith, C. G.

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[CrossRef] [PubMed]

Galbraith, J. A.

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[CrossRef] [PubMed]

Geissbühler, S.

Gillette, J. M.

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[CrossRef] [PubMed]

Girirajan, T. P. K.

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

Gosse, J. A.

T. J. Gould, M. S. Gunewardene, M. V. Gudheti, V. V. Verkhusha, S.-R. Yin, J. A. Gosse, and S. T. Hess, “Nanoscale imaging of molecular positions and anisotropies,” Nat. Methods 5(12), 1027–1030 (2008).
[CrossRef] [PubMed]

Gould, T. J.

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods 5(6), 527–529 (2008).
[CrossRef] [PubMed]

T. J. Gould, M. S. Gunewardene, M. V. Gudheti, V. V. Verkhusha, S.-R. Yin, J. A. Gosse, and S. T. Hess, “Nanoscale imaging of molecular positions and anisotropies,” Nat. Methods 5(12), 1027–1030 (2008).
[CrossRef] [PubMed]

Greengard, A.

S. R. P. Pavani, A. Greengard, and R. Piestun, “Three-dimensional localization with nanometer accuracy using a detector-limited double-helix point spread function system,” Appl. Phys. Lett. 95(2), 021103 (2009).
[CrossRef]

A. Greengard, Y. Y. Schechner, and R. Piestun, “Depth from diffracted rotation,” Opt. Lett. 31(2), 181–183 (2006).
[CrossRef] [PubMed]

Gudheti, M. V.

T. J. Gould, M. S. Gunewardene, M. V. Gudheti, V. V. Verkhusha, S.-R. Yin, J. A. Gosse, and S. T. Hess, “Nanoscale imaging of molecular positions and anisotropies,” Nat. Methods 5(12), 1027–1030 (2008).
[CrossRef] [PubMed]

Gunewardene, M. S.

T. J. Gould, M. S. Gunewardene, M. V. Gudheti, V. V. Verkhusha, S.-R. Yin, J. A. Gosse, and S. T. Hess, “Nanoscale imaging of molecular positions and anisotropies,” Nat. Methods 5(12), 1027–1030 (2008).
[CrossRef] [PubMed]

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]

Ha, T.

T. Ha, T. Enderle, D. S. Chemla, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77(19), 3979–3982 (1996).
[CrossRef] [PubMed]

Hecht, B.

B. Sick, B. Hecht, and L. Novotny, “Orientational imaging of single molecules by annular illumination,” Phys. Rev. Lett. 85(21), 4482–4485 (2000).
[CrossRef] [PubMed]

Hell, S. W.

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]

Hess, H. F.

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[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,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Hess, S. T.

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods 5(6), 527–529 (2008).
[CrossRef] [PubMed]

T. J. Gould, M. S. Gunewardene, M. V. Gudheti, V. V. Verkhusha, S.-R. Yin, J. A. Gosse, and S. T. Hess, “Nanoscale imaging of molecular positions and anisotropies,” Nat. Methods 5(12), 1027–1030 (2008).
[CrossRef] [PubMed]

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

Huang, B.

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319(5864), 810–813 (2008).
[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]

Juette, M. F.

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods 5(6), 527–529 (2008).
[CrossRef] [PubMed]

Kanchanawong, P.

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[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]

Lasser, T.

Lessard, M. D.

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods 5(6), 527–529 (2008).
[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,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Lippincott-Schwartz, J.

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[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,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

G. H. Patterson and J. Lippincott-Schwartz, “A photoactivatable GFP for selective photolabeling of proteins and cells,” Science 297(5588), 1873–1877 (2002).
[CrossRef] [PubMed]

Liu, N.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

Lord, S. J.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

Manley, S.

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[CrossRef] [PubMed]

Märki, I.

Mason, M. D.

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

Mlodzianoski, M. J.

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods 5(6), 527–529 (2008).
[CrossRef] [PubMed]

Moerner, W. E.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

R. M. Dickson, D. J. Norris, and W. E. Moerner, “Simultaneous Imaging of Individual Molecules Aligned Both Parallel and Perpendicular to the Optic Axis,” Phys. Rev. Lett. 81(24), 5322–5325 (1998).
[CrossRef]

Nagpure, B. S.

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods 5(6), 527–529 (2008).
[CrossRef] [PubMed]

Norris, D. J.

R. M. Dickson, D. J. Norris, and W. E. Moerner, “Simultaneous Imaging of Individual Molecules Aligned Both Parallel and Perpendicular to the Optic Axis,” Phys. Rev. Lett. 81(24), 5322–5325 (1998).
[CrossRef]

Novotny, L.

B. Sick, B. Hecht, and L. Novotny, “Orientational imaging of single molecules by annular illumination,” Phys. Rev. Lett. 85(21), 4482–4485 (2000).
[CrossRef] [PubMed]

Ober, R. J.

P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, “Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions,” IEEE Trans. Nanobioscience 3(4), 237–242 (2004).
[CrossRef]

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]

G. H. Patterson and J. Lippincott-Schwartz, “A photoactivatable GFP for selective photolabeling of proteins and cells,” Science 297(5588), 1873–1877 (2002).
[CrossRef] [PubMed]

Pavani, S. R. P.

S. R. P. Pavani, A. Greengard, and R. Piestun, “Three-dimensional localization with nanometer accuracy using a detector-limited double-helix point spread function system,” Appl. Phys. Lett. 95(2), 021103 (2009).
[CrossRef]

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

S. R. P. Pavani and R. Piestun, “High-efficiency rotating point spread functions,” Opt. Express 16(5), 3484–3489 (2008).
[CrossRef] [PubMed]

S. R. P. Pavani and R. Piestun, “Three dimensional tracking of fluorescent microparticles using a photon-limited double-helix response system,” Opt. Express 16(26), 22048–22057 (2008).
[CrossRef] [PubMed]

Piestun, R.

S. R. P. Pavani, A. Greengard, and R. Piestun, “Three-dimensional localization with nanometer accuracy using a detector-limited double-helix point spread function system,” Appl. Phys. Lett. 95(2), 021103 (2009).
[CrossRef]

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

S. R. P. Pavani and R. Piestun, “Three dimensional tracking of fluorescent microparticles using a photon-limited double-helix response system,” Opt. Express 16(26), 22048–22057 (2008).
[CrossRef] [PubMed]

S. R. P. Pavani and R. Piestun, “High-efficiency rotating point spread functions,” Opt. Express 16(5), 3484–3489 (2008).
[CrossRef] [PubMed]

A. Greengard, Y. Y. Schechner, and R. Piestun, “Depth from diffracted rotation,” Opt. Lett. 31(2), 181–183 (2006).
[CrossRef] [PubMed]

Prabhat, P.

P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, “Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions,” IEEE Trans. Nanobioscience 3(4), 237–242 (2004).
[CrossRef]

Ram, S.

P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, “Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions,” IEEE Trans. Nanobioscience 3(4), 237–242 (2004).
[CrossRef]

Romero, C. M.

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]

Schechner, Y. Y.

Shank, C. V.

A. Vaziri, J. Tang, H. Shroff, and C. V. Shank, “Multilayer three-dimensional super resolution imaging of thick biological samples,” Proc. Natl. Acad. Sci. U.S.A. 105(51), 20221–20226 (2008).
[CrossRef] [PubMed]

Shroff, H.

A. Vaziri, J. Tang, H. Shroff, and C. V. Shank, “Multilayer three-dimensional super resolution imaging of thick biological samples,” Proc. Natl. Acad. Sci. U.S.A. 105(51), 20221–20226 (2008).
[CrossRef] [PubMed]

Shtengel, G.

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[CrossRef] [PubMed]

Sick, B.

B. Sick, B. Hecht, and L. Novotny, “Orientational imaging of single molecules by annular illumination,” Phys. Rev. Lett. 85(21), 4482–4485 (2000).
[CrossRef] [PubMed]

Sougrat, R.

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[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,” Science 313(5793), 1642–1645 (2006).
[CrossRef] [PubMed]

Tang, J.

A. Vaziri, J. Tang, H. Shroff, and C. V. Shank, “Multilayer three-dimensional super resolution imaging of thick biological samples,” Proc. Natl. Acad. Sci. U.S.A. 105(51), 20221–20226 (2008).
[CrossRef] [PubMed]

Thompson, M. A.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

Török, P.

Twieg, R. J.

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

Unser, M.

Vaziri, A.

A. Vaziri, J. Tang, H. Shroff, and C. V. Shank, “Multilayer three-dimensional super resolution imaging of thick biological samples,” Proc. Natl. Acad. Sci. U.S.A. 105(51), 20221–20226 (2008).
[CrossRef] [PubMed]

Verkhusha, V. V.

T. J. Gould, M. S. Gunewardene, M. V. Gudheti, V. V. Verkhusha, S.-R. Yin, J. A. Gosse, and S. T. Hess, “Nanoscale imaging of molecular positions and anisotropies,” Nat. Methods 5(12), 1027–1030 (2008).
[CrossRef] [PubMed]

Wang, W.

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319(5864), 810–813 (2008).
[CrossRef] [PubMed]

Ward, E. S.

P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, “Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions,” IEEE Trans. Nanobioscience 3(4), 237–242 (2004).
[CrossRef]

Waterman, C. M.

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[CrossRef] [PubMed]

Weiss, S.

T. Ha, T. Enderle, D. S. Chemla, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77(19), 3979–3982 (1996).
[CrossRef] [PubMed]

Yin, S.-R.

T. J. Gould, M. S. Gunewardene, M. V. Gudheti, V. V. Verkhusha, S.-R. Yin, J. A. Gosse, and S. T. Hess, “Nanoscale imaging of molecular positions and anisotropies,” Nat. Methods 5(12), 1027–1030 (2008).
[CrossRef] [PubMed]

Zhuang, X.

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319(5864), 810–813 (2008).
[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]

Appl. Phys. Lett.

S. R. P. Pavani, A. Greengard, and R. Piestun, “Three-dimensional localization with nanometer accuracy using a detector-limited double-helix point spread function system,” Appl. Phys. Lett. 95(2), 021103 (2009).
[CrossRef]

Biophys. J.

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

IEEE Trans. Nanobioscience

P. Prabhat, S. Ram, E. S. Ward, and R. J. Ober, “Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions,” IEEE Trans. Nanobioscience 3(4), 237–242 (2004).
[CrossRef]

J. Microsc.

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]

Nat. Methods

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]

T. J. Gould, M. S. Gunewardene, M. V. Gudheti, V. V. Verkhusha, S.-R. Yin, J. A. Gosse, and S. T. Hess, “Nanoscale imaging of molecular positions and anisotropies,” Nat. Methods 5(12), 1027–1030 (2008).
[CrossRef] [PubMed]

M. F. Juette, T. J. Gould, M. D. Lessard, M. J. Mlodzianoski, B. S. Nagpure, B. T. Bennett, S. T. Hess, and J. Bewersdorf, “Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples,” Nat. Methods 5(6), 527–529 (2008).
[CrossRef] [PubMed]

Opt. Express

Opt. Lett.

Phys. Rev. Lett.

T. Ha, T. Enderle, D. S. Chemla, and S. Weiss, “Single molecule dynamics studied by polarization modulation,” Phys. Rev. Lett. 77(19), 3979–3982 (1996).
[CrossRef] [PubMed]

R. M. Dickson, D. J. Norris, and W. E. Moerner, “Simultaneous Imaging of Individual Molecules Aligned Both Parallel and Perpendicular to the Optic Axis,” Phys. Rev. Lett. 81(24), 5322–5325 (1998).
[CrossRef]

B. Sick, B. Hecht, and L. Novotny, “Orientational imaging of single molecules by annular illumination,” Phys. Rev. Lett. 85(21), 4482–4485 (2000).
[CrossRef] [PubMed]

Proc. Natl. Acad. Sci. U.S.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]

A. Vaziri, J. Tang, H. Shroff, and C. V. Shank, “Multilayer three-dimensional super resolution imaging of thick biological samples,” Proc. Natl. Acad. Sci. U.S.A. 105(51), 20221–20226 (2008).
[CrossRef] [PubMed]

G. Shtengel, J. A. Galbraith, C. G. Galbraith, J. Lippincott-Schwartz, J. M. Gillette, S. Manley, R. Sougrat, C. M. Waterman, P. Kanchanawong, M. W. Davidson, R. D. Fetter, and H. F. Hess, “Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 3125–3130 (2009).
[CrossRef] [PubMed]

S. R. P. Pavani, M. A. Thompson, J. S. Biteen, S. J. Lord, N. Liu, R. J. Twieg, R. Piestun, and W. E. Moerner, “Three-dimensional, single-molecule fluorescence imaging beyond the diffraction limit by using a double-helix point spread function,” Proc. Natl. Acad. Sci. U.S.A. 106(9), 2995–2999 (2009).
[CrossRef] [PubMed]

Science

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]

B. Huang, W. Wang, M. Bates, and X. Zhuang, “Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy,” Science 319(5864), 810–813 (2008).
[CrossRef] [PubMed]

G. H. Patterson and J. Lippincott-Schwartz, “A photoactivatable GFP for selective photolabeling of proteins and cells,” Science 297(5588), 1873–1877 (2002).
[CrossRef] [PubMed]

Other

S. M. Kay, Fundamentals of Statistical Signal Processing: Estimation Theory (Prentice-Hall, 1993).

A. Yariv, and P. Yeh, Photonics: Optical Electronics in Modern Communications (Oxford University Press, New York, 2007).

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Figures (7)

Fig. 1
Fig. 1

Experimental setup for polarization sensitive 3D superresolution imaging with the double-helix point spread function (DH-PSF). PtK1 cells expressing PA-GFP-tubulin are excited with the 488nm argon laser line and are photoactivated with a 405nm laser diode. The imaging path consists of two polarization channels that are modulated with a spatial light modulator (SLM) encoding the DH-PSF phase mask (bottom right inset). The inset in the middle shows PtK1 microtubules imaged in the horizontal and vertical channels with the standard PSF (SLM off) and with the DH-PSF (SLM loaded with DH-PSF mask). M refers to mirrors; QWP is a quarter wave plate; LW is a 75mm wide-field lens; LO is a 1.3NA objective; LT is a 150mm achromatic lens; LS1, LS2, and LS3 are 250mm achromatic lenses; PBS is a broadband polarization beam splitter; HWP is a broadband half wave plate; EMCCD is an electron multiplying charge coupled device detector.

Fig. 2
Fig. 2

Plots of DH-PSF rotation angle in the (a) horizontal and the (b) vertical channels obtained by moving a 40nm fluorescent microsphere in the axial dimension with 100nm steps. The DH-PSF rotates in opposite direction in the two channels. Scale bar: 1μm

Fig. 3
Fig. 3

(a) Gain in position localization precision obtained by optimally combining position information from the two polarization channels. (b) 3D position histograms of a fluorescent bead showing improved precisions when the horizontal (XH, YH, ZH) and the vertical (XV, YV, ZV) channel estimates are combined (XC, YC, ZC). σH(X,​  Y,Z)=   (2.6,​ ​    2.5,   6.1)nm ; σV(X,​  Y,Z)=(2.5,​ ​    2,5.6)nm ; σC(X,​  Y,Z)=(2,​ ​  1.8,4.5)nm .

Fig. 4
Fig. 4

Position localization histograms of a (a) single PA-GFP molecule in the (b) X, (c) Y, and (d) Z dimensions. σPAGFP(X,​  Y,Z)=   (13.2nm,​ ​    20.3nm,42.8nm) . The average number of detected photons per image is only 246 photons. Scale bar: 1μm

Fig. 5
Fig. 5

Polarization specific 3D superresolution images of a PtK1 cell obtained from (a) horizontal and (b) vertical polarization channels. The superresolution images from the two channels reveal polarization specific features of the cell. For example, the edge of the cell (indicated by cyan dotted arrows) is more prominent in the horizontal than the vertical polarization channel. Scale bars: 1μm

Fig. 6
Fig. 6

X and Y corrections of horizontal and the vertical channels due to aberration-induced systematic shifts in the midpoint of the two PSF lobes.

Fig. 7
Fig. 7

(a) Differential Interference Contrast (DIC) image of PtK1 cells, (b) Cross-polarized image of the same cells as in (a) showing negligible birefringence. Scale bars: 10μm

Equations (13)

Equations on this page are rendered with MathJax. Learn more.

PC   =   αPH   +   βPV,
α   =   σV2σH2+   σV2     ;       β   =   σH2σH2+   σV2,
PG       =       min(σH,   σV)      σC       =       min(σH,   σV)        α2σH2   +   β2σV2   .
σC=       σS   =   σHσVσH2+   σV2.
σC       =       α2σH2   +   (1α)2σV2   ,
σcα=     2ασH2   2(1α)σV22α2σH2+   (1α)2σV2     =     0.
α   =   σV2σH2+   σV2     ;         β   =   σH2σH2+   σV2.
σC   =   σHσVσH2+   σV2.
σH   =   σSκ   ;           σV   =   σS1κ   .
σS   =   σV1κ     =   σV1   σS2σH2.
σS   =   σHσVσH2+   σV2.
CRBC=   CRBH   CRBVCRBH   +   CRBV,
CRBT=   1FH+   FV=     CRBH   CRBVCRBH   +   CRBV.

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