Abstract

As a wide-field imaging technique, super-resolution localization microscopy (SRLM) is theoretically capable of increasing field-of-view (FOV) without sacrificing either imaging speed or spatial resolution. There are two key factors for realizing large FOV SRLM: one is high-power illumination over the whole FOV with sufficient illumination homogeneity and the other is large FOV signal detection by a camera that has large number of pixels and sufficient detection sensitivity. However nowadays, even though the state-of-art scientific complementary metal-oxide semiconductor (sCMOS) cameras provide single molecule fluorescence signal detection ability over an FOV of more than 200 μm × 200 μm, large FOV SRLM still has not been achieved due to the lack of high-power homogeneous illumination. In this paper, we report large FOV SRLM with a high-power homogeneous illumination system. We demonstrate experimentally that our illumination system, which is based on a newly designed multimode fiber combiner, is capable of providing sufficient illumination intensity (~4.7 kW/cm2 @ 640 nm) and excellent illumination homogeneity. Compared with the reported approaches, our illumination system is advantageous in laser power scaling and square-shape illumination without beam clipping. As a result, our system makes full use of the sensor of a representative Hamamatsu Flash 4.0 V2 sCMOS camera (2048 × 2048 active pixels) and achieves a FOV as large as 221 μm × 221 μm with homogeneous spatial resolution. The flexible solution for realizing large FOV SRLM reported in this paper pushes a significant step toward the development of SRLM.

© 2017 Optical Society of America

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2016 (3)

M. de la Roche, Y. Asano, and G. M. Griffiths, “Origins of the cytolytic synapse,” Nat. Rev. Immunol. 16(7), 421–432 (2016).
[Crossref] [PubMed]

K. M. Douglass, C. Sieben, A. Archetti, A. Lambert, and S. Manley, “Super-resolution imaging of multiple cells by optimised flat-field epi-illumination,” Nat. Photonics 10(11), 705–708 (2016).
[Crossref] [PubMed]

J. Deschamps, A. Rowald, and J. Ries, “Efficient homogeneous illumination and optical sectioning for quantitative single-molecule localization microscopy,” Opt. Express 24(24), 28080–28090 (2016).
[Crossref] [PubMed]

2015 (1)

Y. M. Sigal, C. M. Speer, H. P. Babcock, and X. Zhuang, “Mapping synaptic input fields of neurons with super-resolution imaging,” Cell 163(2), 493–505 (2015).
[Crossref] [PubMed]

2014 (3)

M. Gunkel, B. Flottmann, M. Heilemann, J. Reymann, and H. Erfle, “Integrated and correlative high-throughput and super-resolution microscopy,” Histochem. Cell Biol. 141(6), 597–603 (2014).
[Crossref] [PubMed]

S. J. Holden, T. Pengo, K. L. Meibom, C. Fernandez Fernandez, J. Collier, and S. Manley, “High throughput 3D super-resolution microscopy reveals Caulobacter crescentus in vivo Z-ring organization,” Proc. Natl. Acad. Sci. U.S.A. 111(12), 4566–4571 (2014).
[Crossref] [PubMed]

T. Klein, S. Proppert, and M. Sauer, “Eight years of single-molecule localization microscopy,” Histochem. Cell Biol. 141(6), 561–575 (2014).
[Crossref] [PubMed]

2013 (3)

J. Rossy, S. V. Pageon, D. M. Davis, and K. Gaus, “Super-resolution microscopy of the immunological synapse,” Curr. Opin. Immunol. 25(3), 307–312 (2013).
[Crossref] [PubMed]

G. T. Dempsey, “A user’s guide to localization-based super-resolution fluorescence imaging,” Methods Cell Biol. 114, 561–592 (2013).
[Crossref] [PubMed]

F. Huang, T. M. P. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[Crossref] [PubMed]

2012 (3)

2011 (3)

Z. L. Huang, H. Zhu, F. Long, H. Ma, L. Qin, Y. Liu, J. Ding, Z. Zhang, Q. Luo, and S. Zeng, “Localization-based super-resolution microscopy with an sCMOS camera,” Opt. Express 19(20), 19156–19168 (2011).
[Crossref] [PubMed]

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]

F. Schuberts, A. Hoben, K. Bakhshpour, and C. Provost, “Square fibers solve multiple application challenges,” Photon. Spectra 45, 38–41 (2011).

2010 (2)

T. Quan, P. Li, F. Long, S. Zeng, Q. Luo, P. N. Hedde, G. U. Nienhaus, and Z. L. Huang, “Ultra-fast, high-precision image analysis for localization-based super resolution microscopy,” Opt. Express 18(11), 11867–11876 (2010).
[Crossref] [PubMed]

T. Quan, S. Zeng, and Z. L. Huang, “Localization capability and limitation of electron-multiplying charge-coupled, scientific complementary metal-oxide semiconductor, and charge-coupled devices for superresolution imaging,” J. Biomed. Opt. 15(6), 066005 (2010).
[Crossref] [PubMed]

2009 (1)

2008 (1)

M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, and M. Sauer, “Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes,” Angew. Chem. Int. Ed. Engl. 47(33), 6172–6176 (2008).
[Crossref] [PubMed]

2006 (3)

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), 793–795 (2006).
[Crossref] [PubMed]

M. Bigas, E. Cabruja, J. Forest, and J. Salvi, “Review of CMOS image sensors,” Microelectronics J. 37(5), 433–451 (2006).
[Crossref]

2002 (1)

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[Crossref] [PubMed]

Archetti, A.

K. M. Douglass, C. Sieben, A. Archetti, A. Lambert, and S. Manley, “Super-resolution imaging of multiple cells by optimised flat-field epi-illumination,” Nat. Photonics 10(11), 705–708 (2016).
[Crossref] [PubMed]

Asano, Y.

M. de la Roche, Y. Asano, and G. M. Griffiths, “Origins of the cytolytic synapse,” Nat. Rev. Immunol. 16(7), 421–432 (2016).
[Crossref] [PubMed]

Babcock, H. P.

Y. M. Sigal, C. M. Speer, H. P. Babcock, and X. Zhuang, “Mapping synaptic input fields of neurons with super-resolution imaging,” Cell 163(2), 493–505 (2015).
[Crossref] [PubMed]

Baird, M. A.

F. Huang, T. M. P. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[Crossref] [PubMed]

Bakhshpour, K.

F. Schuberts, A. Hoben, K. Bakhshpour, and C. Provost, “Square fibers solve multiple application challenges,” Photon. Spectra 45, 38–41 (2011).

Bates, M.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (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]

Bewersdorf, J.

F. Huang, T. M. P. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[Crossref] [PubMed]

Bigas, M.

M. Bigas, E. Cabruja, J. Forest, and J. Salvi, “Review of CMOS image sensors,” Microelectronics J. 37(5), 433–451 (2006).
[Crossref]

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]

Bruchez, M. P.

Cabruja, E.

M. Bigas, E. Cabruja, J. Forest, and J. Salvi, “Review of CMOS image sensors,” Microelectronics J. 37(5), 433–451 (2006).
[Crossref]

Collier, J.

S. J. Holden, T. Pengo, K. L. Meibom, C. Fernandez Fernandez, J. Collier, and S. Manley, “High throughput 3D super-resolution microscopy reveals Caulobacter crescentus in vivo Z-ring organization,” Proc. Natl. Acad. Sci. U.S.A. 111(12), 4566–4571 (2014).
[Crossref] [PubMed]

Davidson, M. W.

F. Huang, T. M. P. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[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]

Davis, D. M.

J. Rossy, S. V. Pageon, D. M. Davis, and K. Gaus, “Super-resolution microscopy of the immunological synapse,” Curr. Opin. Immunol. 25(3), 307–312 (2013).
[Crossref] [PubMed]

de la Roche, M.

M. de la Roche, Y. Asano, and G. M. Griffiths, “Origins of the cytolytic synapse,” Nat. Rev. Immunol. 16(7), 421–432 (2016).
[Crossref] [PubMed]

Dempsey, G. T.

G. T. Dempsey, “A user’s guide to localization-based super-resolution fluorescence imaging,” Methods Cell Biol. 114, 561–592 (2013).
[Crossref] [PubMed]

Deschamps, J.

Ding, J.

Douglass, K. M.

K. M. Douglass, C. Sieben, A. Archetti, A. Lambert, and S. Manley, “Super-resolution imaging of multiple cells by optimised flat-field epi-illumination,” Nat. Photonics 10(11), 705–708 (2016).
[Crossref] [PubMed]

Duim, W. C.

F. Huang, T. M. P. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[Crossref] [PubMed]

Erfle, H.

M. Gunkel, B. Flottmann, M. Heilemann, J. Reymann, and H. Erfle, “Integrated and correlative high-throughput and super-resolution microscopy,” Histochem. Cell Biol. 141(6), 597–603 (2014).
[Crossref] [PubMed]

Fernandez Fernandez, C.

S. J. Holden, T. Pengo, K. L. Meibom, C. Fernandez Fernandez, J. Collier, and S. Manley, “High throughput 3D super-resolution microscopy reveals Caulobacter crescentus in vivo Z-ring organization,” Proc. Natl. Acad. Sci. U.S.A. 111(12), 4566–4571 (2014).
[Crossref] [PubMed]

Flottmann, B.

M. Gunkel, B. Flottmann, M. Heilemann, J. Reymann, and H. Erfle, “Integrated and correlative high-throughput and super-resolution microscopy,” Histochem. Cell Biol. 141(6), 597–603 (2014).
[Crossref] [PubMed]

Forest, J.

M. Bigas, E. Cabruja, J. Forest, and J. Salvi, “Review of CMOS image sensors,” Microelectronics J. 37(5), 433–451 (2006).
[Crossref]

Gaus, K.

J. Rossy, S. V. Pageon, D. M. Davis, and K. Gaus, “Super-resolution microscopy of the immunological synapse,” Curr. Opin. Immunol. 25(3), 307–312 (2013).
[Crossref] [PubMed]

Griffiths, G. M.

M. de la Roche, Y. Asano, and G. M. Griffiths, “Origins of the cytolytic synapse,” Nat. Rev. Immunol. 16(7), 421–432 (2016).
[Crossref] [PubMed]

Gunkel, M.

M. Gunkel, B. Flottmann, M. Heilemann, J. Reymann, and H. Erfle, “Integrated and correlative high-throughput and super-resolution microscopy,” Histochem. Cell Biol. 141(6), 597–603 (2014).
[Crossref] [PubMed]

Ha, W.

Hartwich, T. M. P.

F. Huang, T. M. P. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[Crossref] [PubMed]

Hedde, P. N.

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.

M. Gunkel, B. Flottmann, M. Heilemann, J. Reymann, and H. Erfle, “Integrated and correlative high-throughput and super-resolution microscopy,” Histochem. Cell Biol. 141(6), 597–603 (2014).
[Crossref] [PubMed]

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]

M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, and M. Sauer, “Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes,” Angew. Chem. Int. Ed. Engl. 47(33), 6172–6176 (2008).
[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]

Hoben, A.

F. Schuberts, A. Hoben, K. Bakhshpour, and C. Provost, “Square fibers solve multiple application challenges,” Photon. Spectra 45, 38–41 (2011).

Holden, S. J.

S. J. Holden, T. Pengo, K. L. Meibom, C. Fernandez Fernandez, J. Collier, and S. Manley, “High throughput 3D super-resolution microscopy reveals Caulobacter crescentus in vivo Z-ring organization,” Proc. Natl. Acad. Sci. U.S.A. 111(12), 4566–4571 (2014).
[Crossref] [PubMed]

Huang, F.

F. Huang, T. M. P. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[Crossref] [PubMed]

Huang, Z. L.

Jung, Y.

Kasper, R.

M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, and M. Sauer, “Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes,” Angew. Chem. Int. Ed. Engl. 47(33), 6172–6176 (2008).
[Crossref] [PubMed]

Kim, J. K.

Klein, T.

T. Klein, S. Proppert, and M. Sauer, “Eight years of single-molecule localization microscopy,” Histochem. Cell Biol. 141(6), 561–575 (2014).
[Crossref] [PubMed]

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]

Lambert, A.

K. M. Douglass, C. Sieben, A. Archetti, A. Lambert, and S. Manley, “Super-resolution imaging of multiple cells by optimised flat-field epi-illumination,” Nat. Photonics 10(11), 705–708 (2016).
[Crossref] [PubMed]

Larson, D. R.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[Crossref] [PubMed]

Lee, S.

Li, P.

Lin, Y.

F. Huang, T. M. P. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (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]

Liu, Y.

Long, F.

Long, J. J.

F. Huang, T. M. P. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[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]

Luo, Q.

Ma, H.

Maji, S.

Manley, S.

K. M. Douglass, C. Sieben, A. Archetti, A. Lambert, and S. Manley, “Super-resolution imaging of multiple cells by optimised flat-field epi-illumination,” Nat. Photonics 10(11), 705–708 (2016).
[Crossref] [PubMed]

S. J. Holden, T. Pengo, K. L. Meibom, C. Fernandez Fernandez, J. Collier, and S. Manley, “High throughput 3D super-resolution microscopy reveals Caulobacter crescentus in vivo Z-ring organization,” Proc. Natl. Acad. Sci. U.S.A. 111(12), 4566–4571 (2014).
[Crossref] [PubMed]

Mehta, D. S.

Meibom, K. L.

S. J. Holden, T. Pengo, K. L. Meibom, C. Fernandez Fernandez, J. Collier, and S. Manley, “High throughput 3D super-resolution microscopy reveals Caulobacter crescentus in vivo Z-ring organization,” Proc. Natl. Acad. Sci. U.S.A. 111(12), 4566–4571 (2014).
[Crossref] [PubMed]

Mothes, W.

F. Huang, T. M. P. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[Crossref] [PubMed]

Mukherjee, A.

M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, and M. Sauer, “Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes,” Angew. Chem. Int. Ed. Engl. 47(33), 6172–6176 (2008).
[Crossref] [PubMed]

Myers, J. R.

F. Huang, T. M. P. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[Crossref] [PubMed]

Naik, D. N.

Nienhaus, G. U.

Oh, K.

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]

Pageon, S. V.

J. Rossy, S. V. Pageon, D. M. Davis, and K. Gaus, “Super-resolution microscopy of the immunological synapse,” Curr. Opin. Immunol. 25(3), 307–312 (2013).
[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]

Pengo, T.

S. J. Holden, T. Pengo, K. L. Meibom, C. Fernandez Fernandez, J. Collier, and S. Manley, “High throughput 3D super-resolution microscopy reveals Caulobacter crescentus in vivo Z-ring organization,” Proc. Natl. Acad. Sci. U.S.A. 111(12), 4566–4571 (2014).
[Crossref] [PubMed]

Proppert, S.

T. Klein, S. Proppert, and M. Sauer, “Eight years of single-molecule localization microscopy,” Histochem. Cell Biol. 141(6), 561–575 (2014).
[Crossref] [PubMed]

Provost, C.

F. Schuberts, A. Hoben, K. Bakhshpour, and C. Provost, “Square fibers solve multiple application challenges,” Photon. Spectra 45, 38–41 (2011).

Qin, L.

Quan, T.

T. Quan, P. Li, F. Long, S. Zeng, Q. Luo, P. N. Hedde, G. U. Nienhaus, and Z. L. Huang, “Ultra-fast, high-precision image analysis for localization-based super resolution microscopy,” Opt. Express 18(11), 11867–11876 (2010).
[Crossref] [PubMed]

T. Quan, S. Zeng, and Z. L. Huang, “Localization capability and limitation of electron-multiplying charge-coupled, scientific complementary metal-oxide semiconductor, and charge-coupled devices for superresolution imaging,” J. Biomed. Opt. 15(6), 066005 (2010).
[Crossref] [PubMed]

Reymann, J.

M. Gunkel, B. Flottmann, M. Heilemann, J. Reymann, and H. Erfle, “Integrated and correlative high-throughput and super-resolution microscopy,” Histochem. Cell Biol. 141(6), 597–603 (2014).
[Crossref] [PubMed]

Ries, J.

Rivera-Molina, F. E.

F. Huang, T. M. P. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[Crossref] [PubMed]

Rossy, J.

J. Rossy, S. V. Pageon, D. M. Davis, and K. Gaus, “Super-resolution microscopy of the immunological synapse,” Curr. Opin. Immunol. 25(3), 307–312 (2013).
[Crossref] [PubMed]

Rowald, A.

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), 793–795 (2006).
[Crossref] [PubMed]

Salvi, J.

M. Bigas, E. Cabruja, J. Forest, and J. Salvi, “Review of CMOS image sensors,” Microelectronics J. 37(5), 433–451 (2006).
[Crossref]

Sauer, M.

T. Klein, S. Proppert, and M. Sauer, “Eight years of single-molecule localization microscopy,” Histochem. Cell Biol. 141(6), 561–575 (2014).
[Crossref] [PubMed]

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]

M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, and M. Sauer, “Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes,” Angew. Chem. Int. Ed. Engl. 47(33), 6172–6176 (2008).
[Crossref] [PubMed]

Saurabh, S.

Schuberts, F.

F. Schuberts, A. Hoben, K. Bakhshpour, and C. Provost, “Square fibers solve multiple application challenges,” Photon. Spectra 45, 38–41 (2011).

Schüttpelz, M.

M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, and M. Sauer, “Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes,” Angew. Chem. Int. Ed. Engl. 47(33), 6172–6176 (2008).
[Crossref] [PubMed]

Seefeldt, B.

M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, and M. Sauer, “Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes,” Angew. Chem. Int. Ed. Engl. 47(33), 6172–6176 (2008).
[Crossref] [PubMed]

Sieben, C.

K. M. Douglass, C. Sieben, A. Archetti, A. Lambert, and S. Manley, “Super-resolution imaging of multiple cells by optimised flat-field epi-illumination,” Nat. Photonics 10(11), 705–708 (2016).
[Crossref] [PubMed]

Sigal, Y. M.

Y. M. Sigal, C. M. Speer, H. P. Babcock, and X. Zhuang, “Mapping synaptic input fields of neurons with super-resolution imaging,” Cell 163(2), 493–505 (2015).
[Crossref] [PubMed]

Singh, R. K.

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]

Speer, C. M.

Y. M. Sigal, C. M. Speer, H. P. Babcock, and X. Zhuang, “Mapping synaptic input fields of neurons with super-resolution imaging,” Cell 163(2), 493–505 (2015).
[Crossref] [PubMed]

Takeda, M.

Thompson, R. E.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[Crossref] [PubMed]

Tinnefeld, P.

M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, and M. Sauer, “Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes,” Angew. Chem. Int. Ed. Engl. 47(33), 6172–6176 (2008).
[Crossref] [PubMed]

Toomre, D.

F. Huang, T. M. P. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[Crossref] [PubMed]

Uchil, P. D.

F. Huang, T. M. P. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[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]

M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, and M. Sauer, “Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes,” Angew. Chem. Int. Ed. Engl. 47(33), 6172–6176 (2008).
[Crossref] [PubMed]

Webb, W. W.

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[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]

Zeng, S.

Zhang, Z.

Zhu, H.

Zhuang, X.

Y. M. Sigal, C. M. Speer, H. P. Babcock, and X. Zhuang, “Mapping synaptic input fields of neurons with super-resolution imaging,” Cell 163(2), 493–505 (2015).
[Crossref] [PubMed]

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
[Crossref] [PubMed]

Angew. Chem. Int. Ed. Engl. (1)

M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld, and M. Sauer, “Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes,” Angew. Chem. Int. Ed. Engl. 47(33), 6172–6176 (2008).
[Crossref] [PubMed]

Appl. Opt. (1)

Biophys. J. (1)

R. E. Thompson, D. R. Larson, and W. W. Webb, “Precise nanometer localization analysis for individual fluorescent probes,” Biophys. J. 82(5), 2775–2783 (2002).
[Crossref] [PubMed]

Cell (1)

Y. M. Sigal, C. M. Speer, H. P. Babcock, and X. Zhuang, “Mapping synaptic input fields of neurons with super-resolution imaging,” Cell 163(2), 493–505 (2015).
[Crossref] [PubMed]

Curr. Opin. Immunol. (1)

J. Rossy, S. V. Pageon, D. M. Davis, and K. Gaus, “Super-resolution microscopy of the immunological synapse,” Curr. Opin. Immunol. 25(3), 307–312 (2013).
[Crossref] [PubMed]

Histochem. Cell Biol. (2)

M. Gunkel, B. Flottmann, M. Heilemann, J. Reymann, and H. Erfle, “Integrated and correlative high-throughput and super-resolution microscopy,” Histochem. Cell Biol. 141(6), 597–603 (2014).
[Crossref] [PubMed]

T. Klein, S. Proppert, and M. Sauer, “Eight years of single-molecule localization microscopy,” Histochem. Cell Biol. 141(6), 561–575 (2014).
[Crossref] [PubMed]

J. Biomed. Opt. (1)

T. Quan, S. Zeng, and Z. L. Huang, “Localization capability and limitation of electron-multiplying charge-coupled, scientific complementary metal-oxide semiconductor, and charge-coupled devices for superresolution imaging,” J. Biomed. Opt. 15(6), 066005 (2010).
[Crossref] [PubMed]

Methods Cell Biol. (1)

G. T. Dempsey, “A user’s guide to localization-based super-resolution fluorescence imaging,” Methods Cell Biol. 114, 561–592 (2013).
[Crossref] [PubMed]

Microelectronics J. (1)

M. Bigas, E. Cabruja, J. Forest, and J. Salvi, “Review of CMOS image sensors,” Microelectronics J. 37(5), 433–451 (2006).
[Crossref]

Nat. Methods (2)

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–795 (2006).
[Crossref] [PubMed]

F. Huang, T. M. P. Hartwich, F. E. Rivera-Molina, Y. Lin, W. C. Duim, J. J. Long, P. D. Uchil, J. R. Myers, M. A. Baird, W. Mothes, M. W. Davidson, D. Toomre, and J. Bewersdorf, “Video-rate nanoscopy using sCMOS camera-specific single-molecule localization algorithms,” Nat. Methods 10(7), 653–658 (2013).
[Crossref] [PubMed]

Nat. Photonics (1)

K. M. Douglass, C. Sieben, A. Archetti, A. Lambert, and S. Manley, “Super-resolution imaging of multiple cells by optimised flat-field epi-illumination,” Nat. Photonics 10(11), 705–708 (2016).
[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]

Nat. Rev. Immunol. (1)

M. de la Roche, Y. Asano, and G. M. Griffiths, “Origins of the cytolytic synapse,” Nat. Rev. Immunol. 16(7), 421–432 (2016).
[Crossref] [PubMed]

Opt. Express (6)

Photon. Spectra (1)

F. Schuberts, A. Hoben, K. Bakhshpour, and C. Provost, “Square fibers solve multiple application challenges,” Photon. Spectra 45, 38–41 (2011).

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

S. J. Holden, T. Pengo, K. L. Meibom, C. Fernandez Fernandez, J. Collier, and S. Manley, “High throughput 3D super-resolution microscopy reveals Caulobacter crescentus in vivo Z-ring organization,” Proc. Natl. Acad. Sci. U.S.A. 111(12), 4566–4571 (2014).
[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 (2)

P. Seitz and A. J. Theuwissen, Single-Photon Imaging (Springer).

J. W. Goodman, Speckle Phenomena in Optics: Theory and Applications (Ben Roberts & Company).

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Figures (10)

Fig. 1
Fig. 1

A schematic diagram (a) and photography (b) of the fiber combiner. The fibers in Part 1 are- the same type of multimode fibers: 50 μm core diameter, 125 μm cladding diameter, and NA = 0.20 (Corning); The fiber in Part 3: 200 μm × 200 μm square core, 420 μm cladding diameter, NA = 0.22 (CeramOptec); FBT: fused biconical taper.

Fig. 2
Fig. 2

The optical setup for testing the illumination homogeneity at the exit of the fiber combiner. Laser: 639 nm (Genesis MX-639-2000, Coherent); SH: Electronic shutter (UNIBLITZ VS14, Vincent Associates); ND: Neutral density filter (NDC-50C-2M, Thorlabs); M1-M2: Aluminum mirrors (BB1-E02, Thorlabs); Iris-Iris2: Iris diaphragms (ID25, Thorlabs); L1: Fiber coupler with a focusing lens (f = 11 mm, SMA connector, PAF-SMA-11-A, Thorlabs); L2: Microscope objective (Plan 10X/NA0.25, Olympus); L3: Achromatic doublet lens (f = 100 mm, AC254-100-A, Thorlabs); sCMOS: Flash 4.0 V2 sCMOS camera (Hamamatsu).

Fig. 3
Fig. 3

The optical setup for quantifying the illumination homogeneity at the sample plane. M1-M7: Aluminum mirrors (Thorlabs); Iris1-Iris4: Iris diaphragms (ID25, Thorlabs); L1/L2: Fiber coupler with a focusing lens (f = 11 mm, PAF-SMA-11-A, Thorlabs); L3: Microscope objective (10 × /NA0.25, Olympus); L4: Achromatic doublet lens (f = 400 mm, AC508-400-A, Thorlabs); L5: Achromatic doublet lens (f = 35 mm, AC254-35-A, Thorlabs); L6: Achromatic doublet lens (f = 150 mm, AC508-150A-ML, Thorlabs); L7: Achromatic doublet lens (f = 300 mm, AC508-300-A, Thorlabs); TL: Tube lens; Objective: Water-immersion microscope objective (60XW/NA1.2, Olympus); DM: Dichroic mirror (ZT405/488/561/647rpc, Chroma); F: Emission filter (ZET405/473/561/640m, Chroma); sCMOS: sCMOS camera (Flash 4.0 V2, Hamamatsu). Pos 1 – Pos 6 indicate the positions used for measuring laser power. Laser 1/Laser 2: 405 nm laser from CNILaser (MLL-III-405); 473 nm laser from CNILaser (MLL-III-473); 561 nm laser from Coherent (Genesis CX-561-3000); 639 nm laser from Coherent (Genesis MX-639-2000); 640 nm laser from LaserWave (LWRL640-3W).

Fig. 4
Fig. 4

(a) The speckle contrast as a function of the rotational speed of the vibration motor. The illumination area was averaged over three data sets with 10 consecutive frames in each data set. The exposure time of the sCMOS camera was set to be 20 ms; (b) The dependence of the speckle contrast on the camera exposure time for several rotational speeds.

Fig. 5
Fig. 5

The illumination homogeneity at the exit of the fiber combiner. The line intensity profiles in (c) correspond to the green lines in (a-b). The calculation was based on single frames and a 639 nm laser (Genesis MX-639-2000, Coherent) was used. Scale bar: 300 μm.

Fig. 6
Fig. 6

Characterizing the illumination homogeneity at the sample plane using fluorescent solution. (a-d) 405 nm; (e-f) 639 nm; (g-h) the combined laser (639 nm and 640 nm); The vibration motor is turned off for (a-b), and turned on for (c-h). The line intensity profiles in the bottom correspond to the images on the top, horizontally along the center of the illumination area. The calculation was based on single frames. Scale bar: 50 μm.

Fig. 7
Fig. 7

The distributions of the speckle contrast for different camera exposure times. All pixels in the horizontal centerline of the illumination area are used for statistics. The statistics were from 500 successive frames of AB9 solution. The excitation laser was 639 nm (Genesis MX-639-2000, Coherent).

Fig. 8
Fig. 8

Characterizing the illumination homogeneity at the sample plane using fluorescent beads. (a) A schematic diagram showing the positions of the fluorescent beads; (b) A representative fluorescence image for the fluorescent beads in position 1; (c) Normalized fluorescence intensities according to the intensity in position 1. The fluorescence intensities are summed over all the signal area for each beads; (d) Localization precision of the same beads in different positions. The total fluorescence intensities and the localization precision were averaged over 500 successive fluorescence images. Scale bar: 50 μm.

Fig. 9
Fig. 9

Homogeneous super-resolution imaging of COS-7 cells using the full sensor of the sCMOS camera. (a) Super-resolution image of COS-7 cells where microtubules were labelled with Alexa Fluor 647; The FOV is 221 μm × 221 μm; (b) Magnified views of the regions with color boxes marked in (a); (c) Pixel intensity profiles through the marked lines in (b) with rectangular box through a microtubule. Red curves are fits to two Gaussian curves. (d) Pixel intensity profiles through the marked lines in (b) with circular box through two adjacent structures. Scale bar: (a) 50 μm, (b) 3 μm.

Fig. 10
Fig. 10

Characterizing the illumination homogeneity across a FOV of 221 μm × 221 μm. (a-b) Fluorescence intensity image and distribution of AB9 solution. The image and the statistics are both from a single image frame; (c) Single molecule fluorescence intensity distribution of Alexa Fluor 647 in COS-7 cells. A maximum likelihood estimator called MaLiang [25] was used to calculate the fluorescence intensities from individual Alexa Fluor 647 molecules, and a total of 10000 successive raw images were used; (d) Normalized single molecule fluorescence intensity distribution inside several small area marked in (c). Scale bar: 50 μm.

Tables (2)

Tables Icon

Table 1 Transmission efficiency of the lasers with different wavelengths a

Tables Icon

Table 2 Transmission efficiency of the combined laser.

Equations (1)

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C= I 2 I 2 I = σ 1 I

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