Abstract

Spinning-disk confocal microscopy is a proven technology for investigating 3D structures of biological specimens. Here we report a super-resolution method based on spinning-disk confocal microscopy that optically improves lateral resolution by a factor of 1.37 with a single exposure. Moreover, deconvolution yields twofold improvement over the diffraction limit. With the help of newly modified Nipkow disk which comprises pinholes and micro-lenses on the front and back respectively, emitted photons from specimen can be optically reassigned to the most probable locations they originate from. Consequently, the improvement in resolution is achieved preserving inherent sectioning capabilities of confocal microscopy. This extremely simple implementation will enable reliable observations at super high resolution in biomedical routine research.

© 2015 Optical Society of America

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2015 (1)

S. Hayashi and Y. Okada, “Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics,” Mol. Biol. Cell 26(9), 1743–1751 (2015).
[Crossref] [PubMed]

2014 (3)

2013 (7)

C. J. R. Sheppard, S. B. Mehta, and R. Heintzmann, “Superresolution by image scanning microscopy using pixel reassignment,” Opt. Lett. 38(15), 2889–2892 (2013).
[Crossref] [PubMed]

G. M. De Luca, R. M. Breedijk, R. A. Brandt, C. H. Zeelenberg, B. E. de Jong, W. Timmermans, L. N. Azar, R. A. Hoebe, S. Stallinga, and E. M. Manders, “Re-scan confocal microscopy: scanning twice for better resolution,” Biomed. Opt. Express 4(11), 2644–2656 (2013).
[Crossref] [PubMed]

O. Schulz, C. Pieper, M. Clever, J. Pfaff, A. Ruhlandt, R. H. Kehlenbach, F. S. Wouters, J. Großhans, G. Bunt, and J. Enderlein, “Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy,” Proc. Natl. Acad. Sci. U.S.A. 110(52), 21000–21005 (2013).
[Crossref] [PubMed]

S. Roth, C. J. R. Sheppard, K. Wicker, and R. Heintzmann, “Optical photon reassignment microscopy (OPRA),” Opt. Nanosc. 2(1), 5 (2013).
[Crossref]

A. G. York, P. Chandris, D. D. Nogare, J. Head, P. Wawrzusin, R. S. Fischer, A. Chitnis, and H. Shroff, “Instant super-resolution imaging in live cells and embryos via analog image processing,” Nat. Methods 10(11), 1122–1126 (2013).
[Crossref] [PubMed]

T. Shimozawa, K. Yamagata, T. Kondo, S. Hayashi, A. Shitamukai, D. Konno, F. Matsuzaki, J. Takayama, S. Onami, H. Nakayama, Y. Kosugi, T. M. Watanabe, K. Fujita, and Y. Mimori-Kiyosue, “Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging,” Proc. Natl. Acad. Sci. U.S.A. 110(9), 3399–3404 (2013).
[Crossref] [PubMed]

N. A. Hosny, M. Song, J. T. Connelly, S. Ameer-Beg, M. M. Knight, and A. P. Wheeler, “Super-resolution imaging strategies for cell biologists using a spinning disk microscope,” PLoS ONE 8(10), e74604 (2013).
[Crossref] [PubMed]

2012 (1)

A. G. York, S. H. Parekh, D. D. 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]

2010 (1)

C. B. Müller and J. Enderlein, “Image scanning microscopy,” Phys. Rev. Lett. 104(19), 198101 (2010).
[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, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006).
[Crossref] [PubMed]

2004 (1)

G. Cox and C. J. R. Sheppard, “Practical limits of resolution in confocal and non-linear microscopy,” Microsc. Res. Tech. 63(1), 18–22 (2004).
[Crossref] [PubMed]

2000 (1)

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]

1994 (1)

1988 (1)

C. J. R. Sheppard, “Super-resolution in confocal imaging,” Optik (Stuttg.) 80(2), 53–54 (1988).

Ameer-Beg, S.

N. A. Hosny, M. Song, J. T. Connelly, S. Ameer-Beg, M. M. Knight, and A. P. Wheeler, “Super-resolution imaging strategies for cell biologists using a spinning disk microscope,” PLoS ONE 8(10), e74604 (2013).
[Crossref] [PubMed]

Azar, L. N.

Bates, M.

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

Betzig, E.

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

Bonifacino, J. S.

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

Brandt, R. A.

Breedijk, R. M.

Brooker, G.

Bunt, G.

O. Schulz, C. Pieper, M. Clever, J. Pfaff, A. Ruhlandt, R. H. Kehlenbach, F. S. Wouters, J. Großhans, G. Bunt, and J. Enderlein, “Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy,” Proc. Natl. Acad. Sci. U.S.A. 110(52), 21000–21005 (2013).
[Crossref] [PubMed]

Chandris, P.

A. G. York, P. Chandris, D. D. Nogare, J. Head, P. Wawrzusin, R. S. Fischer, A. Chitnis, and H. Shroff, “Instant super-resolution imaging in live cells and embryos via analog image processing,” Nat. Methods 10(11), 1122–1126 (2013).
[Crossref] [PubMed]

Chitnis, A.

P. W. Winter, A. G. York, D. D. Nogare, M. Ingaramo, R. Christensen, A. Chitnis, G. H. Patterson, and H. Shroff, “Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples,” Optica 1(3), 181–191 (2014).
[Crossref] [PubMed]

A. G. York, P. Chandris, D. D. Nogare, J. Head, P. Wawrzusin, R. S. Fischer, A. Chitnis, and H. Shroff, “Instant super-resolution imaging in live cells and embryos via analog image processing,” Nat. Methods 10(11), 1122–1126 (2013).
[Crossref] [PubMed]

Chitnis, A. B.

A. G. York, S. H. Parekh, D. D. 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]

Christensen, R.

Clever, M.

O. Schulz, C. Pieper, M. Clever, J. Pfaff, A. Ruhlandt, R. H. Kehlenbach, F. S. Wouters, J. Großhans, G. Bunt, and J. Enderlein, “Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy,” Proc. Natl. Acad. Sci. U.S.A. 110(52), 21000–21005 (2013).
[Crossref] [PubMed]

Combs, C. A.

A. G. York, S. H. Parekh, D. D. 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]

Connelly, J. T.

N. A. Hosny, M. Song, J. T. Connelly, S. Ameer-Beg, M. M. Knight, and A. P. Wheeler, “Super-resolution imaging strategies for cell biologists using a spinning disk microscope,” PLoS ONE 8(10), e74604 (2013).
[Crossref] [PubMed]

Cox, G.

G. Cox and C. J. R. Sheppard, “Practical limits of resolution in confocal and non-linear microscopy,” Microsc. Res. Tech. 63(1), 18–22 (2004).
[Crossref] [PubMed]

Davidson, M. W.

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

de Jong, B. E.

De Luca, G. M.

Enderlein, J.

O. Schulz, C. Pieper, M. Clever, J. Pfaff, A. Ruhlandt, R. H. Kehlenbach, F. S. Wouters, J. Großhans, G. Bunt, and J. Enderlein, “Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy,” Proc. Natl. Acad. Sci. U.S.A. 110(52), 21000–21005 (2013).
[Crossref] [PubMed]

C. B. Müller and J. Enderlein, “Image scanning microscopy,” Phys. Rev. Lett. 104(19), 198101 (2010).
[Crossref] [PubMed]

Fischer, R. S.

A. G. York, P. Chandris, D. D. Nogare, J. Head, P. Wawrzusin, R. S. Fischer, A. Chitnis, and H. Shroff, “Instant super-resolution imaging in live cells and embryos via analog image processing,” Nat. Methods 10(11), 1122–1126 (2013).
[Crossref] [PubMed]

A. G. York, S. H. Parekh, D. D. 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]

Fujita, K.

T. Shimozawa, K. Yamagata, T. Kondo, S. Hayashi, A. Shitamukai, D. Konno, F. Matsuzaki, J. Takayama, S. Onami, H. Nakayama, Y. Kosugi, T. M. Watanabe, K. Fujita, and Y. Mimori-Kiyosue, “Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging,” Proc. Natl. Acad. Sci. U.S.A. 110(9), 3399–3404 (2013).
[Crossref] [PubMed]

Großhans, J.

O. Schulz, C. Pieper, M. Clever, J. Pfaff, A. Ruhlandt, R. H. Kehlenbach, F. S. Wouters, J. Großhans, G. Bunt, and J. Enderlein, “Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy,” Proc. Natl. Acad. Sci. U.S.A. 110(52), 21000–21005 (2013).
[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]

Hayashi, S.

S. Hayashi and Y. Okada, “Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics,” Mol. Biol. Cell 26(9), 1743–1751 (2015).
[Crossref] [PubMed]

T. Shimozawa, K. Yamagata, T. Kondo, S. Hayashi, A. Shitamukai, D. Konno, F. Matsuzaki, J. Takayama, S. Onami, H. Nakayama, Y. Kosugi, T. M. Watanabe, K. Fujita, and Y. Mimori-Kiyosue, “Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging,” Proc. Natl. Acad. Sci. U.S.A. 110(9), 3399–3404 (2013).
[Crossref] [PubMed]

Head, J.

A. G. York, P. Chandris, D. D. Nogare, J. Head, P. Wawrzusin, R. S. Fischer, A. Chitnis, and H. Shroff, “Instant super-resolution imaging in live cells and embryos via analog image processing,” Nat. Methods 10(11), 1122–1126 (2013).
[Crossref] [PubMed]

Heintzmann, R.

S. Roth, C. J. R. Sheppard, K. Wicker, and R. Heintzmann, “Optical photon reassignment microscopy (OPRA),” Opt. Nanosc. 2(1), 5 (2013).
[Crossref]

C. J. R. Sheppard, S. B. Mehta, and R. Heintzmann, “Superresolution by image scanning microscopy using pixel reassignment,” Opt. Lett. 38(15), 2889–2892 (2013).
[Crossref] [PubMed]

Hell, S. W.

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]

Hoebe, R. A.

Hong, A.

M. Ingaramo, A. G. York, P. Wawrzusin, O. Milberg, A. Hong, R. Weigert, H. Shroff, and G. H. Patterson, “Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue,” Proc. Natl. Acad. Sci. U.S.A. 111(14), 5254–5259 (2014).
[Crossref] [PubMed]

Hosny, N. A.

N. A. Hosny, M. Song, J. T. Connelly, S. Ameer-Beg, M. M. Knight, and A. P. Wheeler, “Super-resolution imaging strategies for cell biologists using a spinning disk microscope,” PLoS ONE 8(10), e74604 (2013).
[Crossref] [PubMed]

Ingaramo, M.

M. Ingaramo, A. G. York, P. Wawrzusin, O. Milberg, A. Hong, R. Weigert, H. Shroff, and G. H. Patterson, “Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue,” Proc. Natl. Acad. Sci. U.S.A. 111(14), 5254–5259 (2014).
[Crossref] [PubMed]

P. W. Winter, A. G. York, D. D. Nogare, M. Ingaramo, R. Christensen, A. Chitnis, G. H. Patterson, and H. Shroff, “Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples,” Optica 1(3), 181–191 (2014).
[Crossref] [PubMed]

Kehlenbach, R. H.

O. Schulz, C. Pieper, M. Clever, J. Pfaff, A. Ruhlandt, R. H. Kehlenbach, F. S. Wouters, J. Großhans, G. Bunt, and J. Enderlein, “Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy,” Proc. Natl. Acad. Sci. U.S.A. 110(52), 21000–21005 (2013).
[Crossref] [PubMed]

Knight, M. M.

N. A. Hosny, M. Song, J. T. Connelly, S. Ameer-Beg, M. M. Knight, and A. P. Wheeler, “Super-resolution imaging strategies for cell biologists using a spinning disk microscope,” PLoS ONE 8(10), e74604 (2013).
[Crossref] [PubMed]

Kondo, T.

T. Shimozawa, K. Yamagata, T. Kondo, S. Hayashi, A. Shitamukai, D. Konno, F. Matsuzaki, J. Takayama, S. Onami, H. Nakayama, Y. Kosugi, T. M. Watanabe, K. Fujita, and Y. Mimori-Kiyosue, “Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging,” Proc. Natl. Acad. Sci. U.S.A. 110(9), 3399–3404 (2013).
[Crossref] [PubMed]

Konno, D.

T. Shimozawa, K. Yamagata, T. Kondo, S. Hayashi, A. Shitamukai, D. Konno, F. Matsuzaki, J. Takayama, S. Onami, H. Nakayama, Y. Kosugi, T. M. Watanabe, K. Fujita, and Y. Mimori-Kiyosue, “Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging,” Proc. Natl. Acad. Sci. U.S.A. 110(9), 3399–3404 (2013).
[Crossref] [PubMed]

Kosugi, Y.

T. Shimozawa, K. Yamagata, T. Kondo, S. Hayashi, A. Shitamukai, D. Konno, F. Matsuzaki, J. Takayama, S. Onami, H. Nakayama, Y. Kosugi, T. M. Watanabe, K. Fujita, and Y. Mimori-Kiyosue, “Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging,” Proc. Natl. Acad. Sci. U.S.A. 110(9), 3399–3404 (2013).
[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.

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]

Manders, E. M.

Matsuzaki, F.

T. Shimozawa, K. Yamagata, T. Kondo, S. Hayashi, A. Shitamukai, D. Konno, F. Matsuzaki, J. Takayama, S. Onami, H. Nakayama, Y. Kosugi, T. M. Watanabe, K. Fujita, and Y. Mimori-Kiyosue, “Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging,” Proc. Natl. Acad. Sci. U.S.A. 110(9), 3399–3404 (2013).
[Crossref] [PubMed]

Mehta, S. B.

Milberg, O.

M. Ingaramo, A. G. York, P. Wawrzusin, O. Milberg, A. Hong, R. Weigert, H. Shroff, and G. H. Patterson, “Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue,” Proc. Natl. Acad. Sci. U.S.A. 111(14), 5254–5259 (2014).
[Crossref] [PubMed]

Mimori-Kiyosue, Y.

T. Shimozawa, K. Yamagata, T. Kondo, S. Hayashi, A. Shitamukai, D. Konno, F. Matsuzaki, J. Takayama, S. Onami, H. Nakayama, Y. Kosugi, T. M. Watanabe, K. Fujita, and Y. Mimori-Kiyosue, “Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging,” Proc. Natl. Acad. Sci. U.S.A. 110(9), 3399–3404 (2013).
[Crossref] [PubMed]

Mione, M.

A. G. York, S. H. Parekh, D. D. 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]

Müller, C. B.

C. B. Müller and J. Enderlein, “Image scanning microscopy,” Phys. Rev. Lett. 104(19), 198101 (2010).
[Crossref] [PubMed]

Nakayama, H.

T. Shimozawa, K. Yamagata, T. Kondo, S. Hayashi, A. Shitamukai, D. Konno, F. Matsuzaki, J. Takayama, S. Onami, H. Nakayama, Y. Kosugi, T. M. Watanabe, K. Fujita, and Y. Mimori-Kiyosue, “Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging,” Proc. Natl. Acad. Sci. U.S.A. 110(9), 3399–3404 (2013).
[Crossref] [PubMed]

Nogare, D. D.

P. W. Winter, A. G. York, D. D. Nogare, M. Ingaramo, R. Christensen, A. Chitnis, G. H. Patterson, and H. Shroff, “Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples,” Optica 1(3), 181–191 (2014).
[Crossref] [PubMed]

A. G. York, P. Chandris, D. D. Nogare, J. Head, P. Wawrzusin, R. S. Fischer, A. Chitnis, and H. Shroff, “Instant super-resolution imaging in live cells and embryos via analog image processing,” Nat. Methods 10(11), 1122–1126 (2013).
[Crossref] [PubMed]

A. G. York, S. H. Parekh, D. D. 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]

Okada, Y.

S. Hayashi and Y. Okada, “Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics,” Mol. Biol. Cell 26(9), 1743–1751 (2015).
[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]

Onami, S.

T. Shimozawa, K. Yamagata, T. Kondo, S. Hayashi, A. Shitamukai, D. Konno, F. Matsuzaki, J. Takayama, S. Onami, H. Nakayama, Y. Kosugi, T. M. Watanabe, K. Fujita, and Y. Mimori-Kiyosue, “Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging,” Proc. Natl. Acad. Sci. U.S.A. 110(9), 3399–3404 (2013).
[Crossref] [PubMed]

Parekh, S. H.

A. G. York, S. H. Parekh, D. D. 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.

M. Ingaramo, A. G. York, P. Wawrzusin, O. Milberg, A. Hong, R. Weigert, H. Shroff, and G. H. Patterson, “Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue,” Proc. Natl. Acad. Sci. U.S.A. 111(14), 5254–5259 (2014).
[Crossref] [PubMed]

P. W. Winter, A. G. York, D. D. Nogare, M. Ingaramo, R. Christensen, A. Chitnis, G. H. Patterson, and H. Shroff, “Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples,” Optica 1(3), 181–191 (2014).
[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]

Pfaff, J.

O. Schulz, C. Pieper, M. Clever, J. Pfaff, A. Ruhlandt, R. H. Kehlenbach, F. S. Wouters, J. Großhans, G. Bunt, and J. Enderlein, “Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy,” Proc. Natl. Acad. Sci. U.S.A. 110(52), 21000–21005 (2013).
[Crossref] [PubMed]

Pieper, C.

O. Schulz, C. Pieper, M. Clever, J. Pfaff, A. Ruhlandt, R. H. Kehlenbach, F. S. Wouters, J. Großhans, G. Bunt, and J. Enderlein, “Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy,” Proc. Natl. Acad. Sci. U.S.A. 110(52), 21000–21005 (2013).
[Crossref] [PubMed]

Roth, S.

S. Roth, C. J. R. Sheppard, K. Wicker, and R. Heintzmann, “Optical photon reassignment microscopy (OPRA),” Opt. Nanosc. 2(1), 5 (2013).
[Crossref]

Ruhlandt, A.

O. Schulz, C. Pieper, M. Clever, J. Pfaff, A. Ruhlandt, R. H. Kehlenbach, F. S. Wouters, J. Großhans, G. Bunt, and J. Enderlein, “Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy,” Proc. Natl. Acad. Sci. U.S.A. 110(52), 21000–21005 (2013).
[Crossref] [PubMed]

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]

Schulz, O.

O. Schulz, C. Pieper, M. Clever, J. Pfaff, A. Ruhlandt, R. H. Kehlenbach, F. S. Wouters, J. Großhans, G. Bunt, and J. Enderlein, “Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy,” Proc. Natl. Acad. Sci. U.S.A. 110(52), 21000–21005 (2013).
[Crossref] [PubMed]

Sheppard, C. J. R.

S. Roth, C. J. R. Sheppard, K. Wicker, and R. Heintzmann, “Optical photon reassignment microscopy (OPRA),” Opt. Nanosc. 2(1), 5 (2013).
[Crossref]

C. J. R. Sheppard, S. B. Mehta, and R. Heintzmann, “Superresolution by image scanning microscopy using pixel reassignment,” Opt. Lett. 38(15), 2889–2892 (2013).
[Crossref] [PubMed]

G. Cox and C. J. R. Sheppard, “Practical limits of resolution in confocal and non-linear microscopy,” Microsc. Res. Tech. 63(1), 18–22 (2004).
[Crossref] [PubMed]

C. J. R. Sheppard, “Super-resolution in confocal imaging,” Optik (Stuttg.) 80(2), 53–54 (1988).

Shimozawa, T.

T. Shimozawa, K. Yamagata, T. Kondo, S. Hayashi, A. Shitamukai, D. Konno, F. Matsuzaki, J. Takayama, S. Onami, H. Nakayama, Y. Kosugi, T. M. Watanabe, K. Fujita, and Y. Mimori-Kiyosue, “Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging,” Proc. Natl. Acad. Sci. U.S.A. 110(9), 3399–3404 (2013).
[Crossref] [PubMed]

Shitamukai, A.

T. Shimozawa, K. Yamagata, T. Kondo, S. Hayashi, A. Shitamukai, D. Konno, F. Matsuzaki, J. Takayama, S. Onami, H. Nakayama, Y. Kosugi, T. M. Watanabe, K. Fujita, and Y. Mimori-Kiyosue, “Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging,” Proc. Natl. Acad. Sci. U.S.A. 110(9), 3399–3404 (2013).
[Crossref] [PubMed]

Shroff, H.

P. W. Winter, A. G. York, D. D. Nogare, M. Ingaramo, R. Christensen, A. Chitnis, G. H. Patterson, and H. Shroff, “Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples,” Optica 1(3), 181–191 (2014).
[Crossref] [PubMed]

M. Ingaramo, A. G. York, P. Wawrzusin, O. Milberg, A. Hong, R. Weigert, H. Shroff, and G. H. Patterson, “Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue,” Proc. Natl. Acad. Sci. U.S.A. 111(14), 5254–5259 (2014).
[Crossref] [PubMed]

A. G. York, P. Chandris, D. D. Nogare, J. Head, P. Wawrzusin, R. S. Fischer, A. Chitnis, and H. Shroff, “Instant super-resolution imaging in live cells and embryos via analog image processing,” Nat. Methods 10(11), 1122–1126 (2013).
[Crossref] [PubMed]

A. G. York, S. H. Parekh, D. D. 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]

Siegel, N.

Song, M.

N. A. Hosny, M. Song, J. T. Connelly, S. Ameer-Beg, M. M. Knight, and A. P. Wheeler, “Super-resolution imaging strategies for cell biologists using a spinning disk microscope,” PLoS ONE 8(10), e74604 (2013).
[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.

Takayama, J.

T. Shimozawa, K. Yamagata, T. Kondo, S. Hayashi, A. Shitamukai, D. Konno, F. Matsuzaki, J. Takayama, S. Onami, H. Nakayama, Y. Kosugi, T. M. Watanabe, K. Fujita, and Y. Mimori-Kiyosue, “Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging,” Proc. Natl. Acad. Sci. U.S.A. 110(9), 3399–3404 (2013).
[Crossref] [PubMed]

Temprine, K.

A. G. York, S. H. Parekh, D. D. 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]

Timmermans, W.

Watanabe, T. M.

T. Shimozawa, K. Yamagata, T. Kondo, S. Hayashi, A. Shitamukai, D. Konno, F. Matsuzaki, J. Takayama, S. Onami, H. Nakayama, Y. Kosugi, T. M. Watanabe, K. Fujita, and Y. Mimori-Kiyosue, “Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging,” Proc. Natl. Acad. Sci. U.S.A. 110(9), 3399–3404 (2013).
[Crossref] [PubMed]

Wawrzusin, P.

M. Ingaramo, A. G. York, P. Wawrzusin, O. Milberg, A. Hong, R. Weigert, H. Shroff, and G. H. Patterson, “Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue,” Proc. Natl. Acad. Sci. U.S.A. 111(14), 5254–5259 (2014).
[Crossref] [PubMed]

A. G. York, P. Chandris, D. D. Nogare, J. Head, P. Wawrzusin, R. S. Fischer, A. Chitnis, and H. Shroff, “Instant super-resolution imaging in live cells and embryos via analog image processing,” Nat. Methods 10(11), 1122–1126 (2013).
[Crossref] [PubMed]

Weigert, R.

M. Ingaramo, A. G. York, P. Wawrzusin, O. Milberg, A. Hong, R. Weigert, H. Shroff, and G. H. Patterson, “Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue,” Proc. Natl. Acad. Sci. U.S.A. 111(14), 5254–5259 (2014).
[Crossref] [PubMed]

Wheeler, A. P.

N. A. Hosny, M. Song, J. T. Connelly, S. Ameer-Beg, M. M. Knight, and A. P. Wheeler, “Super-resolution imaging strategies for cell biologists using a spinning disk microscope,” PLoS ONE 8(10), e74604 (2013).
[Crossref] [PubMed]

Wichmann, J.

Wicker, K.

S. Roth, C. J. R. Sheppard, K. Wicker, and R. Heintzmann, “Optical photon reassignment microscopy (OPRA),” Opt. Nanosc. 2(1), 5 (2013).
[Crossref]

Winter, P. W.

Wouters, F. S.

O. Schulz, C. Pieper, M. Clever, J. Pfaff, A. Ruhlandt, R. H. Kehlenbach, F. S. Wouters, J. Großhans, G. Bunt, and J. Enderlein, “Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy,” Proc. Natl. Acad. Sci. U.S.A. 110(52), 21000–21005 (2013).
[Crossref] [PubMed]

Yamagata, K.

T. Shimozawa, K. Yamagata, T. Kondo, S. Hayashi, A. Shitamukai, D. Konno, F. Matsuzaki, J. Takayama, S. Onami, H. Nakayama, Y. Kosugi, T. M. Watanabe, K. Fujita, and Y. Mimori-Kiyosue, “Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging,” Proc. Natl. Acad. Sci. U.S.A. 110(9), 3399–3404 (2013).
[Crossref] [PubMed]

York, A. G.

P. W. Winter, A. G. York, D. D. Nogare, M. Ingaramo, R. Christensen, A. Chitnis, G. H. Patterson, and H. Shroff, “Two-photon instant structured illumination microscopy improves the depth penetration of super-resolution imaging in thick scattering samples,” Optica 1(3), 181–191 (2014).
[Crossref] [PubMed]

M. Ingaramo, A. G. York, P. Wawrzusin, O. Milberg, A. Hong, R. Weigert, H. Shroff, and G. H. Patterson, “Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue,” Proc. Natl. Acad. Sci. U.S.A. 111(14), 5254–5259 (2014).
[Crossref] [PubMed]

A. G. York, P. Chandris, D. D. Nogare, J. Head, P. Wawrzusin, R. S. Fischer, A. Chitnis, and H. Shroff, “Instant super-resolution imaging in live cells and embryos via analog image processing,” Nat. Methods 10(11), 1122–1126 (2013).
[Crossref] [PubMed]

A. G. York, S. H. Parekh, D. D. 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]

Zeelenberg, C. H.

Zhuang, X.

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

Biomed. Opt. Express (1)

J. Microsc. (1)

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]

Microsc. Res. Tech. (1)

G. Cox and C. J. R. Sheppard, “Practical limits of resolution in confocal and non-linear microscopy,” Microsc. Res. Tech. 63(1), 18–22 (2004).
[Crossref] [PubMed]

Mol. Biol. Cell (1)

S. Hayashi and Y. Okada, “Ultrafast superresolution fluorescence imaging with spinning disk confocal microscope optics,” Mol. Biol. Cell 26(9), 1743–1751 (2015).
[Crossref] [PubMed]

Nat. Methods (3)

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]

A. G. York, S. H. Parekh, D. D. 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]

A. G. York, P. Chandris, D. D. Nogare, J. Head, P. Wawrzusin, R. S. Fischer, A. Chitnis, and H. Shroff, “Instant super-resolution imaging in live cells and embryos via analog image processing,” Nat. Methods 10(11), 1122–1126 (2013).
[Crossref] [PubMed]

Opt. Express (1)

Opt. Lett. (2)

Opt. Nanosc. (1)

S. Roth, C. J. R. Sheppard, K. Wicker, and R. Heintzmann, “Optical photon reassignment microscopy (OPRA),” Opt. Nanosc. 2(1), 5 (2013).
[Crossref]

Optica (1)

Optik (Stuttg.) (1)

C. J. R. Sheppard, “Super-resolution in confocal imaging,” Optik (Stuttg.) 80(2), 53–54 (1988).

Phys. Rev. Lett. (1)

C. B. Müller and J. Enderlein, “Image scanning microscopy,” Phys. Rev. Lett. 104(19), 198101 (2010).
[Crossref] [PubMed]

PLoS ONE (1)

N. A. Hosny, M. Song, J. T. Connelly, S. Ameer-Beg, M. M. Knight, and A. P. Wheeler, “Super-resolution imaging strategies for cell biologists using a spinning disk microscope,” PLoS ONE 8(10), e74604 (2013).
[Crossref] [PubMed]

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

T. Shimozawa, K. Yamagata, T. Kondo, S. Hayashi, A. Shitamukai, D. Konno, F. Matsuzaki, J. Takayama, S. Onami, H. Nakayama, Y. Kosugi, T. M. Watanabe, K. Fujita, and Y. Mimori-Kiyosue, “Improving spinning disk confocal microscopy by preventing pinhole cross-talk for intravital imaging,” Proc. Natl. Acad. Sci. U.S.A. 110(9), 3399–3404 (2013).
[Crossref] [PubMed]

M. Ingaramo, A. G. York, P. Wawrzusin, O. Milberg, A. Hong, R. Weigert, H. Shroff, and G. H. Patterson, “Two-photon excitation improves multifocal structured illumination microscopy in thick scattering tissue,” Proc. Natl. Acad. Sci. U.S.A. 111(14), 5254–5259 (2014).
[Crossref] [PubMed]

O. Schulz, C. Pieper, M. Clever, J. Pfaff, A. Ruhlandt, R. H. Kehlenbach, F. S. Wouters, J. Großhans, G. Bunt, and J. Enderlein, “Resolution doubling in fluorescence microscopy with confocal spinning-disk image scanning microscopy,” Proc. Natl. Acad. Sci. U.S.A. 110(52), 21000–21005 (2013).
[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 (1)

J. B. Pawley, Handbook of Biological Confocal Microscopy, 3rd ed. (Plenum, 2006).

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Figures (4)

Fig. 1
Fig. 1

Simplified schematic diagram of the optical setup. A micro-lens array (MLA) disk focuses the excitation light onto pinholes on a micro-lens added pinhole array (MLPHA) disk through a dichroic mirror (DM). The excitation light passing through the micro-lenses on the lower surface of the MLPHA is focused onto specimen by an objective lens. The emission collected by the same objective lens is contracted and pinholed by the MLPHA and imaged by the camera through an emission filter (EM). The scanning unit of SD-OPR is implemented on a CSU-X1 spinning-disk confocal microscope by substituting the internal spinning-disk assembly. The scanning unit is attached to an inverted microscope (Olympus®, IX71) using custom-made joint including a tube lens (f = 250 mm). In this arrangement, the original tube lens of the microscope is not on the optical path. The specimen is held on a motorized microscope stage (OptoSigma®, BIOS-225T). A piezo objective scanner (PI®, P-721.10) is used to perform axial scanning. The optical system is built on a vibration isolation table (HERZ, TDI-1585LAB). Although we didn’t use autofocus and environmental chamber in our setup, the use of them may be essential for thermal stability to perform long-term live-cell imaging.

Fig. 2
Fig. 2

Comparison of point spread functions. Subdiffractive fluorescent beads (Thermo Fisher Scientific®, FluoSpheres® F8803, 0.1 μm diameter, yellow-green stained) were imaged with widefield, normal Yokogawa® CSU-X1 spinning-disk (SD) and SD-OPR. Deconvolved images were obtained by applying 3D deconvolution (Wiener filter preconditioned Landweber algorithm) using Piotr Wendykier’s parallel iterative deconvolution plugin for ImageJ. The first and the second image columns show X-Y and X-Z cross sectional views of PSFs respectively. The scale bars represent 0.5 μm.

Fig. 3
Fig. 3

Imaging of microtubules in fixed bovine pulmonary artery endothelial cells. (a) Raw widefield image. (b) Deconvolved widefield image. (c) Raw SD-OPR image. (d) Deconvolved SD-OPR image. The scale bars in the panels a, b, c and d represent 10 μm. Deconvolved images were obtained by applying 3D deconvolution using Huygens® (Scientific Volume Imaging). (e–h) Magnified views of smaller areas corresponding to the rectangles in the panels a, b, c and d. The scale bars in the panels e, f, g and h represent 1 μm. (i) Sectional plots of pixel intensities across the yellow lines in the panels e, f, g and h. (j) Sectional plot of pixel intensities across the red line in the panel h. Fittings of two Gaussian functions are also shown with broken lines, and the distance between the peaks of Gaussian functions is 113 nm.

Fig. 4
Fig. 4

Simulation of excitation focal shift vs. wavelength. Wavelength characteristic of axial shift of excitation focus in the specimen was calculated using Zemax® (Zemax LLC) optical simulation software. To calculate the focal shift caused only by the micro-lenses, aberration-free microscope model was used in the simulation. The axial shift of excitation focus was evaluated in the wavelength range of 405 to 640 nm.

Tables (1)

Tables Icon

Table 1 FWHM measurements of point spread functions.

Equations (6)

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

I(r,s)= d r U(r r +s)E(r r )c( r )
I ISM (r)= ds I(rs/2,s)= d r ( ds U(r r +s/2)E(r r s/2) )c( r ).
I ISM (r)= d r ( 4 dv U(2v)E(2r2 r 2v) )c( r ).
U ISM (r)=4 dv U(2v)E(2r2v).
U ISM (r)=2 π σexp( r 2 / σ 2 ).
U ˜ ISM (k)=2 U ˜ (k/2) E ˜ (k/2)

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