Abstract

Recent advances in superresolution fluorescence microscopy have been limited by a belief that surpassing two-fold resolution enhancement of the Rayleigh resolution limit requires stimulated emission or the fluorophore to undergo state transitions. Here we demonstrate a new superresolution method that requires only image acquisitions with a focused illumination spot and computational post-processing. The proposed method utilizes the focused illumination spot to effectively reduce the object size and enhance the object sparsity and consequently increases the resolution and accuracy through nonlinear image post-processing. This method clearly resolves 70nm resolution test objects emitting ~530nm light with a 1.4 numerical aperture (NA) objective, and, when imaging through a 0.5NA objective, exhibits high spatial frequencies comparable to a 1.4NA widefield image, both demonstrating a resolution enhancement above two-fold of the Rayleigh resolution limit. More importantly, we examine how the resolution increases with photon numbers, and show that the more-than-two-fold enhancement is achievable with realistic photon budgets.

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

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References

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    [Crossref]

2016 (1)

L. Wiemerslage and D. Lee, “Quantification of mitochondrial morphology in neurites of dopaminergic neurons using multiple parameters,” J. Neurosci. Methods 262, 56–65 (2016).
[Crossref] [PubMed]

2013 (2)

N. Banterle, K. H. Bui, E. A. Lemke, and M. Beck, “Fourier ring correlation as a resolution criterion for super-resolution microscopy,” J. Struct. Biol. 183(3), 363–367 (2013).
[Crossref] [PubMed]

R. P. J. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, “Measuring image resolution in optical nanoscopy,” Nat. Methods 10(6), 557–562 (2013).
[Crossref] [PubMed]

2012 (2)

A. G. York, S. H. Parekh, D. Dalle Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods 9(7), 749–754 (2012).
[Crossref] [PubMed]

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A. 109(3), E135–E143 (2012).
[Crossref] [PubMed]

2011 (3)

D. T. Burnette, P. Sengupta, Y. Dai, J. Lippincott-Schwartz, and B. Kachar, “Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules,” Proc. Natl. Acad. Sci. U.S.A. 108(52), 21081–21086 (2011).
[Crossref] [PubMed]

S. R. Becker, E. J. Candès, and M. C. Grant, “Templates for convex cone problems with applications to sparse signal recovery,” Math. Program. Comput. 3(3), 165–218 (2011).
[Crossref]

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[Crossref] [PubMed]

2009 (1)

T. Dertinger, R. Colyer, G. Iyer, S. Weiss, and J. Enderlein, “Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI),” Proc. Natl. Acad. Sci. U.S.A. 106(52), 22287–22292 (2009).
[Crossref] [PubMed]

2008 (1)

N. C. Shaner, M. Z. Lin, M. R. McKeown, P. A. Steinbach, K. L. Hazelwood, M. W. Davidson, and R. Y. Tsien, “Improving the photostability of bright monomeric orange and red fluorescent proteins,” Nat. Methods 5(6), 545–551 (2008).
[Crossref] [PubMed]

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, “Stochastic optical reconstruction microscopy (STORM) provides sub-diffraction-limit image resolution,” Nat. Methods 3(10), 793–795 (2006).
[Crossref] [PubMed]

2000 (2)

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

L. Greenbaum, C. Rothmann, R. Lavie, and Z. Malik, “Green fluorescent protein photobleaching: a model for protein damage by endogenous and exogenous singlet oxygen,” Biol. Chem. 381(12), 1251–1258 (2000).
[Crossref] [PubMed]

1999 (1)

1997 (2)

R. Swaminathan, C. P. Hoang, and A. S. Verkman, “Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion,” Biophys. J. 72(4), 1900–1907 (1997).
[Crossref] [PubMed]

C. J. R. Sheppard and K. G. Larkin, “Vectorial pupil functions and vectorial transfer functions,” Optik (Stuttg.) 107, 79–87 (1997).

1995 (1)

L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, “Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy,” Biophys. J. 68(6), 2588–2600 (1995).
[Crossref] [PubMed]

1994 (1)

1993 (1)

P. J. Sementilli, B. R. Hunt, and M. S. Nadar, “Analysis of the limit to superresolution in incoherent imaging,” J. Opt. Soc. Am. 10(11), 2265–2276 (1993).
[Crossref]

1992 (1)

D. L. Donoho, I. M. Johnstone, J. C. Hoch, and A. S. Stern, “Maximum entropy and the nearly black object,” J. R. Stat. Soc. B 54(1), 41–81 (1992).

1972 (1)

1903 (1)

L. Rayleigh, “On the theory of optical images, with special reference to the microscope,” J. R. Microsc. Soc. 23(4), 447–473 (1903).
[Crossref]

Banterle, N.

N. Banterle, K. H. Bui, E. A. Lemke, and M. Beck, “Fourier ring correlation as a resolution criterion for super-resolution microscopy,” J. Struct. Biol. 183(3), 363–367 (2013).
[Crossref] [PubMed]

Bates, M.

R. P. J. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, “Measuring image resolution in optical nanoscopy,” Nat. Methods 10(6), 557–562 (2013).
[Crossref] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Stochastic optical reconstruction microscopy (STORM) provides sub-diffraction-limit image resolution,” Nat. Methods 3(10), 793–795 (2006).
[Crossref] [PubMed]

Beck, M.

N. Banterle, K. H. Bui, E. A. Lemke, and M. Beck, “Fourier ring correlation as a resolution criterion for super-resolution microscopy,” J. Struct. Biol. 183(3), 363–367 (2013).
[Crossref] [PubMed]

Becker, S. R.

S. R. Becker, E. J. Candès, and M. C. Grant, “Templates for convex cone problems with applications to sparse signal recovery,” Math. Program. Comput. 3(3), 165–218 (2011).
[Crossref]

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]

Bui, K. H.

N. Banterle, K. H. Bui, E. A. Lemke, and M. Beck, “Fourier ring correlation as a resolution criterion for super-resolution microscopy,” J. Struct. Biol. 183(3), 363–367 (2013).
[Crossref] [PubMed]

Burke, J. J.

Burnette, D. T.

D. T. Burnette, P. Sengupta, Y. Dai, J. Lippincott-Schwartz, and B. Kachar, “Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules,” Proc. Natl. Acad. Sci. U.S.A. 108(52), 21081–21086 (2011).
[Crossref] [PubMed]

Candès, E. J.

S. R. Becker, E. J. Candès, and M. C. Grant, “Templates for convex cone problems with applications to sparse signal recovery,” Math. Program. Comput. 3(3), 165–218 (2011).
[Crossref]

Chitnis, A. B.

A. G. York, S. H. Parekh, D. Dalle Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods 9(7), 749–754 (2012).
[Crossref] [PubMed]

Colyer, R.

T. Dertinger, R. Colyer, G. Iyer, S. Weiss, and J. Enderlein, “Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI),” Proc. Natl. Acad. Sci. U.S.A. 106(52), 22287–22292 (2009).
[Crossref] [PubMed]

Combs, C. A.

A. G. York, S. H. Parekh, D. Dalle Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods 9(7), 749–754 (2012).
[Crossref] [PubMed]

Dai, Y.

D. T. Burnette, P. Sengupta, Y. Dai, J. Lippincott-Schwartz, and B. Kachar, “Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules,” Proc. Natl. Acad. Sci. U.S.A. 108(52), 21081–21086 (2011).
[Crossref] [PubMed]

Dalle Nogare, D.

A. G. York, S. H. Parekh, D. Dalle Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods 9(7), 749–754 (2012).
[Crossref] [PubMed]

Davidson, M. W.

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A. 109(3), E135–E143 (2012).
[Crossref] [PubMed]

N. C. Shaner, M. Z. Lin, M. R. McKeown, P. A. Steinbach, K. L. Hazelwood, M. W. Davidson, and R. Y. Tsien, “Improving the photostability of bright monomeric orange and red fluorescent proteins,” Nat. Methods 5(6), 545–551 (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,” Science 313(5793), 1642–1645 (2006).
[Crossref] [PubMed]

Dertinger, T.

T. Dertinger, R. Colyer, G. Iyer, S. Weiss, and J. Enderlein, “Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI),” Proc. Natl. Acad. Sci. U.S.A. 106(52), 22287–22292 (2009).
[Crossref] [PubMed]

Donoho, D. L.

D. L. Donoho, I. M. Johnstone, J. C. Hoch, and A. S. Stern, “Maximum entropy and the nearly black object,” J. R. Stat. Soc. B 54(1), 41–81 (1992).

Enderlein, J.

T. Dertinger, R. Colyer, G. Iyer, S. Weiss, and J. Enderlein, “Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI),” Proc. Natl. Acad. Sci. U.S.A. 106(52), 22287–22292 (2009).
[Crossref] [PubMed]

Fischer, R. S.

A. G. York, S. H. Parekh, D. Dalle Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods 9(7), 749–754 (2012).
[Crossref] [PubMed]

Frieden, B. R.

Grant, M. C.

S. R. Becker, E. J. Candès, and M. C. Grant, “Templates for convex cone problems with applications to sparse signal recovery,” Math. Program. Comput. 3(3), 165–218 (2011).
[Crossref]

Greenbaum, L.

L. Greenbaum, C. Rothmann, R. Lavie, and Z. Malik, “Green fluorescent protein photobleaching: a model for protein damage by endogenous and exogenous singlet oxygen,” Biol. Chem. 381(12), 1251–1258 (2000).
[Crossref] [PubMed]

Grünwald, D.

R. P. J. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, “Measuring image resolution in optical nanoscopy,” Nat. Methods 10(6), 557–562 (2013).
[Crossref] [PubMed]

Gustafsson, M. G.

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

Gustafsson, M. G. L.

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A. 109(3), E135–E143 (2012).
[Crossref] [PubMed]

Hazelwood, K. L.

N. C. Shaner, M. Z. Lin, M. R. McKeown, P. A. Steinbach, K. L. Hazelwood, M. W. Davidson, and R. Y. Tsien, “Improving the photostability of bright monomeric orange and red fluorescent proteins,” Nat. Methods 5(6), 545–551 (2008).
[Crossref] [PubMed]

Heidbreder, M.

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[Crossref] [PubMed]

Heilemann, M.

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[Crossref] [PubMed]

Hell, S. W.

Hennink, E. J.

L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, “Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy,” Biophys. J. 68(6), 2588–2600 (1995).
[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,” Science 313(5793), 1642–1645 (2006).
[Crossref] [PubMed]

Hoang, C. P.

R. Swaminathan, C. P. Hoang, and A. S. Verkman, “Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion,” Biophys. J. 72(4), 1900–1907 (1997).
[Crossref] [PubMed]

Hoch, J. C.

D. L. Donoho, I. M. Johnstone, J. C. Hoch, and A. S. Stern, “Maximum entropy and the nearly black object,” J. R. Stat. Soc. B 54(1), 41–81 (1992).

Hunt, B. R.

P. J. Sementilli, B. R. Hunt, and M. S. Nadar, “Analysis of the limit to superresolution in incoherent imaging,” J. Opt. Soc. Am. 10(11), 2265–2276 (1993).
[Crossref]

Iyer, G.

T. Dertinger, R. Colyer, G. Iyer, S. Weiss, and J. Enderlein, “Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI),” Proc. Natl. Acad. Sci. U.S.A. 106(52), 22287–22292 (2009).
[Crossref] [PubMed]

Johansson, G. A.

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A. 109(3), E135–E143 (2012).
[Crossref] [PubMed]

Johnstone, I. M.

D. L. Donoho, I. M. Johnstone, J. C. Hoch, and A. S. Stern, “Maximum entropy and the nearly black object,” J. R. Stat. Soc. B 54(1), 41–81 (1992).

Kachar, B.

D. T. Burnette, P. Sengupta, Y. Dai, J. Lippincott-Schwartz, and B. Kachar, “Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules,” Proc. Natl. Acad. Sci. U.S.A. 108(52), 21081–21086 (2011).
[Crossref] [PubMed]

Kamps-Hughes, N.

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A. 109(3), E135–E143 (2012).
[Crossref] [PubMed]

Klar, T. A.

Klein, T.

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[Crossref] [PubMed]

Larkin, K. G.

C. J. R. Sheppard and K. G. Larkin, “Vectorial pupil functions and vectorial transfer functions,” Optik (Stuttg.) 107, 79–87 (1997).

Lavie, R.

L. Greenbaum, C. Rothmann, R. Lavie, and Z. Malik, “Green fluorescent protein photobleaching: a model for protein damage by endogenous and exogenous singlet oxygen,” Biol. Chem. 381(12), 1251–1258 (2000).
[Crossref] [PubMed]

Lee, D.

L. Wiemerslage and D. Lee, “Quantification of mitochondrial morphology in neurites of dopaminergic neurons using multiple parameters,” J. Neurosci. Methods 262, 56–65 (2016).
[Crossref] [PubMed]

Lemke, E. A.

N. Banterle, K. H. Bui, E. A. Lemke, and M. Beck, “Fourier ring correlation as a resolution criterion for super-resolution microscopy,” J. Struct. Biol. 183(3), 363–367 (2013).
[Crossref] [PubMed]

Lidke, K. A.

R. P. J. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, “Measuring image resolution in optical nanoscopy,” Nat. Methods 10(6), 557–562 (2013).
[Crossref] [PubMed]

Lin, M. Z.

N. C. Shaner, M. Z. Lin, M. R. McKeown, P. A. Steinbach, K. L. Hazelwood, M. W. Davidson, and R. Y. Tsien, “Improving the photostability of bright monomeric orange and red fluorescent proteins,” Nat. Methods 5(6), 545–551 (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.

D. T. Burnette, P. Sengupta, Y. Dai, J. Lippincott-Schwartz, and B. Kachar, “Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules,” Proc. Natl. Acad. Sci. U.S.A. 108(52), 21081–21086 (2011).
[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]

Löschberger, A.

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[Crossref] [PubMed]

Macklin, J. J.

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A. 109(3), E135–E143 (2012).
[Crossref] [PubMed]

Malik, Z.

L. Greenbaum, C. Rothmann, R. Lavie, and Z. Malik, “Green fluorescent protein photobleaching: a model for protein damage by endogenous and exogenous singlet oxygen,” Biol. Chem. 381(12), 1251–1258 (2000).
[Crossref] [PubMed]

McKeown, M. R.

N. C. Shaner, M. Z. Lin, M. R. McKeown, P. A. Steinbach, K. L. Hazelwood, M. W. Davidson, and R. Y. Tsien, “Improving the photostability of bright monomeric orange and red fluorescent proteins,” Nat. Methods 5(6), 545–551 (2008).
[Crossref] [PubMed]

Mione, M.

A. G. York, S. H. Parekh, D. Dalle Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods 9(7), 749–754 (2012).
[Crossref] [PubMed]

Nadar, M. S.

P. J. Sementilli, B. R. Hunt, and M. S. Nadar, “Analysis of the limit to superresolution in incoherent imaging,” J. Opt. Soc. Am. 10(11), 2265–2276 (1993).
[Crossref]

Nieuwenhuizen, R. P. J.

R. P. J. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, “Measuring image resolution in optical nanoscopy,” Nat. Methods 10(6), 557–562 (2013).
[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,” Science 313(5793), 1642–1645 (2006).
[Crossref] [PubMed]

Parekh, S. H.

A. G. York, S. H. Parekh, D. Dalle Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods 9(7), 749–754 (2012).
[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]

Puig, D. L.

R. P. J. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, “Measuring image resolution in optical nanoscopy,” Nat. Methods 10(6), 557–562 (2013).
[Crossref] [PubMed]

Rayleigh, L.

L. Rayleigh, “On the theory of optical images, with special reference to the microscope,” J. R. Microsc. Soc. 23(4), 447–473 (1903).
[Crossref]

Rego, E. H.

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A. 109(3), E135–E143 (2012).
[Crossref] [PubMed]

Rieger, B.

R. P. J. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, “Measuring image resolution in optical nanoscopy,” Nat. Methods 10(6), 557–562 (2013).
[Crossref] [PubMed]

Rothmann, C.

L. Greenbaum, C. Rothmann, R. Lavie, and Z. Malik, “Green fluorescent protein photobleaching: a model for protein damage by endogenous and exogenous singlet oxygen,” Biol. Chem. 381(12), 1251–1258 (2000).
[Crossref] [PubMed]

Rust, M. J.

M. J. Rust, M. Bates, and X. Zhuang, “Stochastic optical reconstruction microscopy (STORM) provides sub-diffraction-limit image resolution,” Nat. Methods 3(10), 793–795 (2006).
[Crossref] [PubMed]

Sauer, M.

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[Crossref] [PubMed]

Sementilli, P. J.

P. J. Sementilli, B. R. Hunt, and M. S. Nadar, “Analysis of the limit to superresolution in incoherent imaging,” J. Opt. Soc. Am. 10(11), 2265–2276 (1993).
[Crossref]

Sengupta, P.

D. T. Burnette, P. Sengupta, Y. Dai, J. Lippincott-Schwartz, and B. Kachar, “Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules,” Proc. Natl. Acad. Sci. U.S.A. 108(52), 21081–21086 (2011).
[Crossref] [PubMed]

Shaner, N. C.

N. C. Shaner, M. Z. Lin, M. R. McKeown, P. A. Steinbach, K. L. Hazelwood, M. W. Davidson, and R. Y. Tsien, “Improving the photostability of bright monomeric orange and red fluorescent proteins,” Nat. Methods 5(6), 545–551 (2008).
[Crossref] [PubMed]

Shao, L.

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A. 109(3), E135–E143 (2012).
[Crossref] [PubMed]

Sheppard, C. J. R.

C. J. R. Sheppard and K. G. Larkin, “Vectorial pupil functions and vectorial transfer functions,” Optik (Stuttg.) 107, 79–87 (1997).

Shroff, H.

A. G. York, S. H. Parekh, D. Dalle Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods 9(7), 749–754 (2012).
[Crossref] [PubMed]

Song, L.

L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, “Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy,” Biophys. J. 68(6), 2588–2600 (1995).
[Crossref] [PubMed]

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]

Stallinga, S.

R. P. J. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, “Measuring image resolution in optical nanoscopy,” Nat. Methods 10(6), 557–562 (2013).
[Crossref] [PubMed]

Steinbach, P. A.

N. C. Shaner, M. Z. Lin, M. R. McKeown, P. A. Steinbach, K. L. Hazelwood, M. W. Davidson, and R. Y. Tsien, “Improving the photostability of bright monomeric orange and red fluorescent proteins,” Nat. Methods 5(6), 545–551 (2008).
[Crossref] [PubMed]

Stern, A. S.

D. L. Donoho, I. M. Johnstone, J. C. Hoch, and A. S. Stern, “Maximum entropy and the nearly black object,” J. R. Stat. Soc. B 54(1), 41–81 (1992).

Swaminathan, R.

R. Swaminathan, C. P. Hoang, and A. S. Verkman, “Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion,” Biophys. J. 72(4), 1900–1907 (1997).
[Crossref] [PubMed]

Tanke, H. J.

L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, “Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy,” Biophys. J. 68(6), 2588–2600 (1995).
[Crossref] [PubMed]

Temprine, K.

A. G. York, S. H. Parekh, D. Dalle Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods 9(7), 749–754 (2012).
[Crossref] [PubMed]

Tsien, R. Y.

N. C. Shaner, M. Z. Lin, M. R. McKeown, P. A. Steinbach, K. L. Hazelwood, M. W. Davidson, and R. Y. Tsien, “Improving the photostability of bright monomeric orange and red fluorescent proteins,” Nat. Methods 5(6), 545–551 (2008).
[Crossref] [PubMed]

van de Linde, S.

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[Crossref] [PubMed]

Verkman, A. S.

R. Swaminathan, C. P. Hoang, and A. S. Verkman, “Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion,” Biophys. J. 72(4), 1900–1907 (1997).
[Crossref] [PubMed]

Weiss, S.

T. Dertinger, R. Colyer, G. Iyer, S. Weiss, and J. Enderlein, “Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI),” Proc. Natl. Acad. Sci. U.S.A. 106(52), 22287–22292 (2009).
[Crossref] [PubMed]

Wichmann, J.

Wiemerslage, L.

L. Wiemerslage and D. Lee, “Quantification of mitochondrial morphology in neurites of dopaminergic neurons using multiple parameters,” J. Neurosci. Methods 262, 56–65 (2016).
[Crossref] [PubMed]

Winoto, L.

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A. 109(3), E135–E143 (2012).
[Crossref] [PubMed]

Wolter, S.

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[Crossref] [PubMed]

York, A. G.

A. G. York, S. H. Parekh, D. Dalle Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods 9(7), 749–754 (2012).
[Crossref] [PubMed]

Young, I. T.

L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, “Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy,” Biophys. J. 68(6), 2588–2600 (1995).
[Crossref] [PubMed]

Zhuang, X.

M. J. Rust, M. Bates, and X. Zhuang, “Stochastic optical reconstruction microscopy (STORM) provides sub-diffraction-limit image resolution,” Nat. Methods 3(10), 793–795 (2006).
[Crossref] [PubMed]

Biol. Chem. (1)

L. Greenbaum, C. Rothmann, R. Lavie, and Z. Malik, “Green fluorescent protein photobleaching: a model for protein damage by endogenous and exogenous singlet oxygen,” Biol. Chem. 381(12), 1251–1258 (2000).
[Crossref] [PubMed]

Biophys. J. (2)

R. Swaminathan, C. P. Hoang, and A. S. Verkman, “Photobleaching recovery and anisotropy decay of green fluorescent protein GFP-S65T in solution and cells: cytoplasmic viscosity probed by green fluorescent protein translational and rotational diffusion,” Biophys. J. 72(4), 1900–1907 (1997).
[Crossref] [PubMed]

L. Song, E. J. Hennink, I. T. Young, and H. J. Tanke, “Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy,” Biophys. J. 68(6), 2588–2600 (1995).
[Crossref] [PubMed]

J. Microsc. (1)

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

J. Neurosci. Methods (1)

L. Wiemerslage and D. Lee, “Quantification of mitochondrial morphology in neurites of dopaminergic neurons using multiple parameters,” J. Neurosci. Methods 262, 56–65 (2016).
[Crossref] [PubMed]

J. Opt. Soc. Am. (2)

P. J. Sementilli, B. R. Hunt, and M. S. Nadar, “Analysis of the limit to superresolution in incoherent imaging,” J. Opt. Soc. Am. 10(11), 2265–2276 (1993).
[Crossref]

B. R. Frieden and J. J. Burke, “Restoring with maximum entropy, II: superresolution of photographs of diffraction-blurred impulses,” J. Opt. Soc. Am. 62(10), 1202–1210 (1972).
[Crossref]

J. R. Microsc. Soc. (1)

L. Rayleigh, “On the theory of optical images, with special reference to the microscope,” J. R. Microsc. Soc. 23(4), 447–473 (1903).
[Crossref]

J. R. Stat. Soc. B (1)

D. L. Donoho, I. M. Johnstone, J. C. Hoch, and A. S. Stern, “Maximum entropy and the nearly black object,” J. R. Stat. Soc. B 54(1), 41–81 (1992).

J. Struct. Biol. (1)

N. Banterle, K. H. Bui, E. A. Lemke, and M. Beck, “Fourier ring correlation as a resolution criterion for super-resolution microscopy,” J. Struct. Biol. 183(3), 363–367 (2013).
[Crossref] [PubMed]

Math. Program. Comput. (1)

S. R. Becker, E. J. Candès, and M. C. Grant, “Templates for convex cone problems with applications to sparse signal recovery,” Math. Program. Comput. 3(3), 165–218 (2011).
[Crossref]

Nat. Methods (4)

R. P. J. Nieuwenhuizen, K. A. Lidke, M. Bates, D. L. Puig, D. Grünwald, S. Stallinga, and B. Rieger, “Measuring image resolution in optical nanoscopy,” Nat. Methods 10(6), 557–562 (2013).
[Crossref] [PubMed]

A. G. York, S. H. Parekh, D. Dalle Nogare, R. S. Fischer, K. Temprine, M. Mione, A. B. Chitnis, C. A. Combs, and H. Shroff, “Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy,” Nat. Methods 9(7), 749–754 (2012).
[Crossref] [PubMed]

N. C. Shaner, M. Z. Lin, M. R. McKeown, P. A. Steinbach, K. L. Hazelwood, M. W. Davidson, and R. Y. Tsien, “Improving the photostability of bright monomeric orange and red fluorescent proteins,” Nat. Methods 5(6), 545–551 (2008).
[Crossref] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Stochastic optical reconstruction microscopy (STORM) provides sub-diffraction-limit image resolution,” Nat. Methods 3(10), 793–795 (2006).
[Crossref] [PubMed]

Nat. Protoc. (1)

S. van de Linde, A. Löschberger, T. Klein, M. Heidbreder, S. Wolter, M. Heilemann, and M. Sauer, “Direct stochastic optical reconstruction microscopy with standard fluorescent probes,” Nat. Protoc. 6(7), 991–1009 (2011).
[Crossref] [PubMed]

Opt. Lett. (2)

Optik (Stuttg.) (1)

C. J. R. Sheppard and K. G. Larkin, “Vectorial pupil functions and vectorial transfer functions,” Optik (Stuttg.) 107, 79–87 (1997).

Proc. Natl. Acad. Sci. U.S.A. (3)

D. T. Burnette, P. Sengupta, Y. Dai, J. Lippincott-Schwartz, and B. Kachar, “Bleaching/blinking assisted localization microscopy for superresolution imaging using standard fluorescent molecules,” Proc. Natl. Acad. Sci. U.S.A. 108(52), 21081–21086 (2011).
[Crossref] [PubMed]

T. Dertinger, R. Colyer, G. Iyer, S. Weiss, and J. Enderlein, “Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI),” Proc. Natl. Acad. Sci. U.S.A. 106(52), 22287–22292 (2009).
[Crossref] [PubMed]

E. H. Rego, L. Shao, J. J. Macklin, L. Winoto, G. A. Johansson, N. Kamps-Hughes, M. W. Davidson, and M. G. L. Gustafsson, “Nonlinear structured-illumination microscopy with a photoswitchable protein reveals cellular structures at 50-nm resolution,” Proc. Natl. Acad. Sci. U.S.A. 109(3), E135–E143 (2012).
[Crossref] [PubMed]

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]

Other (3)

J. B. Pawley, Handbook of biological confocal microscopy (Springer, third ed., 2006).

J. W. Goodman, Introduction to Fourier Optics (Roberts and Company Publishers, third ed., 2005), Chap 6.5.2.

J. W. Goodman, Introduction to Fourier Optics (Roberts and Company Publishers, third ed., 2005), Chap 6.6.5.

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Figures (8)

Fig. 1
Fig. 1 Resolving two incoherent point sources using NNLS inversions at various point-to-point distances and photon numbers. (a), (b) Simulated image formation where a two-point object convolves with the imaging system PSF to form the acquired images, assuming 1:1 magnification. The ‘*’ sign denotes a convolution operation. Each simulated acquired image in (b) is a representative image out of 403 independent simulations, assuming Poisson noise. (c) The criteria determining whether the two point sources are discernable in a particular restoration: successful discernment is declared only when both the average and maximum values in Region1 and Region2 are greater than those in Region0. The two white dots indicate the positions of the two point sources. (d) NNLS restorations corresponding to acquired images in (b). (e) Discernment rates versus photon numbers at various two-point distances. (f) Estimated number of photons per emitter that are required to achieve a 75% discernment rate for various resolution enhancement, defined as dR/d. See Appendix A for further details.
Fig. 2
Fig. 2 Comparison of NNLS restorations from widefield and focused-spot illuminations. (a) An object consisting of three lines and an overlay of the object and 25 illumination spot positions, denoted by the cyan crosses. The brightness of the middle line is 3/4 of the top/bottom line. (b) Simulated two-dimensional intensity profiles of the illumination spots. (c) The object illuminated by the 25 focused spots individually. (d) Simulated acquired images with individual focused-spot illuminations. (e) Individual NNLS restorations of acquired images in (d), with dDict = dR/12. (f) Sum of restorations in (e). (g) The effective widefield image. (h) NNLS restoration of (g). (i) Definition of the line restoration quality (LRQ) of a particular object line: the average of the values (Avg) within the line region divided by their standard deviations (Std). (j) LRQs versus number of photons for spot illumination and widefield illumination. Due to the symmetry of Region1 and Region3, we average their LRQs in the plot, and denote such an average as LRQ1,3. See Appendix B for further details.
Fig. 3
Fig. 3 Results of two imaging experiments using focused spot illumination in conjunction with NNLS inversions. (a)-(d) Imaging experiment using a 70nm resolution test sample as the object. (e)-(h) Imaging experiment using fluorescently stained mitochondria as the object. (a) Effective widefield image. (b) Restoration from our method, with dDict = dCP/12. (c) Enlarged restoration and widefield image of selected regions. The enlarged widefield images are interpolated using a MATLAB ‘bicubic’ method. (d) One-dimensional intensity profiles of the six DNA origami-fluorescent dye structures. (e) Effective 0.5NA widefield image. (f) Restoration from our method, obtained by summing up the restorations from individual 0.5NA images using focused-spot illumination, with dDict = dCP/3. (g) 1.4NA Widefield image of the same region. (h) Average strengths of the 2D DFTs in (e)-(g) versus spatial frequency radius. See Appendix H for further details for calculating frequency ring average.
Fig. 4
Fig. 4 The resolution enhancement provided by our method shows steady increases with signal-to-noise ratios. (a)-(c) Effective widefield images obtained by summing up the acquired focused-spot illumination images at three excitation laser powers. Objects are fluorescently stained mitochondria in fixed cells. The three laser powers are selected such that the photon counts in the effective widefield images are significantly lower than those can be supported by commonly used fluorophores. (d) Average strengths of widefield image 2D DFTs versus spatial frequency radius. The inset shows an experimentally measured imaging system PSF. (e)-(g) Restorations from our method, obtained by summing up the restorations from individual acquired images that are used to generate effective widefield images (a)-(c) respectively. (h) Correlation coefficients of restoration 2D DFTs versus spatial frequency radius. 1/7 threshold is used to determine the effective cutoff frequencies of the restorations. Appendices G and H provide details for estimating signal-to-noise ratios and frequency ring correlation.
Fig. 5
Fig. 5 Restorations obtained using our method, with various dictionary PSF spacings and signal levels. (a)-(c) Center positions (blue dots) of dictionary PSFs at dDict ≈103.2nm, 51.6nm, or 17.2nm, respectively. The green dashed circle indicates the first dark fringe of a diffraction-limited focused illumination spot. (d)-(f) Effective widefield images at three signal levels. (g)-(o) Restorations obtained using our method with 9 distinct combinations of dDict and the signal level.
Fig. 6
Fig. 6 Illustrations of photon number estimation. (a) The object is assumed to be a 100nm square carrying 10 fluorescent protein molecules. The white squares represent the locations of the 10 molecules. (b) The microscope objective only collects fluorescent photons emitted into its collection angle. Here, Fluorescent Photon 1 and 2 respectively exemplify photons that will or will not be collected by the microscope objective. (c) A computationally simulated widefield image of the object. (d) Widefield image of a fluorescently stained biological sample. (e) Widefield image of a DNA origami-fluorescent dye sample. These two samples have the same fluorophore with different labeling densities. The same amount of illumination is used for acquiring images in (d) and (e).
Fig. 7
Fig. 7 Illustrations of frequency ring average calculation. (a) During image acquisition, one image is acquired at each illumination spot position. (b), (c) Frequency ring average calculations for effective widefield images and our imaging method.
Fig. 8
Fig. 8 Illustrations of frequency ring correlation calculation. (a) During image acquisition, we acquire two images at each illumination position, and randomly assign them to two distinct data sets. (b) Frequency ring correlation calculation.

Equations (2)

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min x, x i 0 i=1 n DictPSF x i I PSFi I Image 2 2 ,
SNR=10lo g 10 Avg( n MaxCounts ) Avg( n MaxCounts )+Avg(SD( n DarkCounts ) ) 2 dB,

Metrics