Abstract

We report a robust two-color method for super-resolution localization microscopy. Two-dye combination of Alexa647 and Alexa750 in an imaging buffer containing COT and using TCEP as switching regent provides matched and balanced switching characteristics for both dyes, allowing simultaneous capture of both on a single camera. Active sample locking stabilizes sample with 1nm accuracy during imaging. With over 4,000 photons emitted from both dyes, two-color superresolution images with high-quality were obtained in a wide range of samples including cell cultures, tissue sections and yeast cells.

© 2015 Optical Society of America

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  1. 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]
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    [Crossref] [PubMed]
  3. S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
    [Crossref] [PubMed]
  4. M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317(5845), 1749–1753 (2007).
    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
  11. J. Tang, J. Akerboom, A. Vaziri, L. L. Looger, and C. V. Shank, “Near-isotropic 3D optical nanoscopy with photon-limited chromophores,” Proc. Natl. Acad. Sci. U.S.A. 107(22), 10068–10073 (2010).
    [Crossref] [PubMed]
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  13. J. C. Vaughan, G. T. Dempsey, E. Sun, and X. Zhuang, “Phosphine Quenching of Cyanine Dyes as a Versatile Tool for Fluorescence Microscopy,” J. Am. Chem. Soc. 135(4), 1197–1200 (2013).
    [Crossref] [PubMed]
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    [Crossref] [PubMed]
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    [Crossref] [PubMed]
  18. A. Dani, B. Huang, J. Bergan, C. Dulac, and X. Zhuang, “Superresolution Imaging of Chemical Synapses in the Brain,” Neuron 68(5), 843–856 (2010).
    [Crossref] [PubMed]
  19. J. Ries, C. Kaplan, E. Platonova, H. Eghlidi, and H. Ewers, “A simple, versatile method for GFP-based super-resolution microscopy via nanobodies,” Nat. Methods 9(6), 582–584 (2012).
    [Crossref] [PubMed]

2014 (2)

J. Tam, G. A. Cordier, J. S. Borbely, Á. Sandoval Álvarez, and M. Lakadamyali, “Cross-Talk-Free Multi-Color STORM Imaging Using a Single Fluorophore,” PLoS ONE 9(7), e101772 (2014).
[Crossref] [PubMed]

R. Jungmann, M. S. Avendaño, J. B. Woehrstein, M. Dai, W. M. Shih, and P. Yin, “Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT,” Nat. Methods 11(3), 313–318 (2014).
[Crossref] [PubMed]

2013 (4)

J. C. Vaughan, G. T. Dempsey, E. Sun, and X. Zhuang, “Phosphine Quenching of Cyanine Dyes as a Versatile Tool for Fluorescence Microscopy,” J. Am. Chem. Soc. 135(4), 1197–1200 (2013).
[Crossref] [PubMed]

N. Olivier, D. Keller, P. Gönczy, and S. Manley, “Resolution Doubling In 3D-STORM Imaging through Improved Buffers,” PLoS ONE 8(7), e69004 (2013).
[Crossref] [PubMed]

R. McGorty, D. Kamiyama, and B. Huang, “Active microscope stabilization in three dimensions using image correlation,” Opt .Nanoscopy 2(1), 3 (2013).
[Crossref] [PubMed]

P. K. Koo, S. U. Setru, and S. G. J. Mochrie, “Active drift stabilization in three dimensions via image cross-correlation,” Rev. Sci. Instrum. 84(10), 103705 (2013).
[Crossref] [PubMed]

2012 (2)

J. Ries, C. Kaplan, E. Platonova, H. Eghlidi, and H. Ewers, “A simple, versatile method for GFP-based super-resolution microscopy via nanobodies,” Nat. Methods 9(6), 582–584 (2012).
[Crossref] [PubMed]

M. Bates, G. T. Dempsey, K. H. Chen, and X. Zhuang, “Multicolor Super-Resolution Fluorescence Imaging via Multi-Parameter Fluorophore Detection,” ChemPhysChem 13(1), 99–107 (2012).
[Crossref] [PubMed]

2011 (1)

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8(12), 1027–1036 (2011).
[Crossref] [PubMed]

2010 (3)

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, “Multicolor Fluorescence Nanoscopy in Fixed and Living Cells by Exciting Conventional Fluorophores with a Single Wavelength,” Biophys. J. 99(8), 2686–2694 (2010).
[Crossref] [PubMed]

J. Tang, J. Akerboom, A. Vaziri, L. L. Looger, and C. V. Shank, “Near-isotropic 3D optical nanoscopy with photon-limited chromophores,” Proc. Natl. Acad. Sci. U.S.A. 107(22), 10068–10073 (2010).
[Crossref] [PubMed]

A. Dani, B. Huang, J. Bergan, C. Dulac, and X. Zhuang, “Superresolution Imaging of Chemical Synapses in the Brain,” Neuron 68(5), 843–856 (2010).
[Crossref] [PubMed]

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]

2007 (2)

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317(5845), 1749–1753 (2007).
[Crossref] [PubMed]

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, “Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes,” Proc. Natl. Acad. Sci. U.S.A. 104(51), 20308–20313 (2007).
[Crossref] [PubMed]

2006 (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]

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]

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[Crossref] [PubMed]

1987 (1)

Akerboom, J.

J. Tang, J. Akerboom, A. Vaziri, L. L. Looger, and C. V. Shank, “Near-isotropic 3D optical nanoscopy with photon-limited chromophores,” Proc. Natl. Acad. Sci. U.S.A. 107(22), 10068–10073 (2010).
[Crossref] [PubMed]

Avendaño, M. S.

R. Jungmann, M. S. Avendaño, J. B. Woehrstein, M. Dai, W. M. Shih, and P. Yin, “Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT,” Nat. Methods 11(3), 313–318 (2014).
[Crossref] [PubMed]

Bates, M.

M. Bates, G. T. Dempsey, K. H. Chen, and X. Zhuang, “Multicolor Super-Resolution Fluorescence Imaging via Multi-Parameter Fluorophore Detection,” ChemPhysChem 13(1), 99–107 (2012).
[Crossref] [PubMed]

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8(12), 1027–1036 (2011).
[Crossref] [PubMed]

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317(5845), 1749–1753 (2007).
[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]

Bergan, J.

A. Dani, B. Huang, J. Bergan, C. Dulac, and X. Zhuang, “Superresolution Imaging of Chemical Synapses in the Brain,” Neuron 68(5), 843–856 (2010).
[Crossref] [PubMed]

Betzig, E.

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, “Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes,” Proc. Natl. Acad. Sci. U.S.A. 104(51), 20308–20313 (2007).
[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]

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]

Borbely, J. S.

J. Tam, G. A. Cordier, J. S. Borbely, Á. Sandoval Álvarez, and M. Lakadamyali, “Cross-Talk-Free Multi-Color STORM Imaging Using a Single Fluorophore,” PLoS ONE 9(7), e101772 (2014).
[Crossref] [PubMed]

Chen, K. H.

M. Bates, G. T. Dempsey, K. H. Chen, and X. Zhuang, “Multicolor Super-Resolution Fluorescence Imaging via Multi-Parameter Fluorophore Detection,” ChemPhysChem 13(1), 99–107 (2012).
[Crossref] [PubMed]

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8(12), 1027–1036 (2011).
[Crossref] [PubMed]

Cordier, G. A.

J. Tam, G. A. Cordier, J. S. Borbely, Á. Sandoval Álvarez, and M. Lakadamyali, “Cross-Talk-Free Multi-Color STORM Imaging Using a Single Fluorophore,” PLoS ONE 9(7), e101772 (2014).
[Crossref] [PubMed]

Dai, M.

R. Jungmann, M. S. Avendaño, J. B. Woehrstein, M. Dai, W. M. Shih, and P. Yin, “Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT,” Nat. Methods 11(3), 313–318 (2014).
[Crossref] [PubMed]

Dani, A.

A. Dani, B. Huang, J. Bergan, C. Dulac, and X. Zhuang, “Superresolution Imaging of Chemical Synapses in the Brain,” Neuron 68(5), 843–856 (2010).
[Crossref] [PubMed]

Davidson, M. W.

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, “Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes,” Proc. Natl. Acad. Sci. U.S.A. 104(51), 20308–20313 (2007).
[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]

Dempsey, G. T.

J. C. Vaughan, G. T. Dempsey, E. Sun, and X. Zhuang, “Phosphine Quenching of Cyanine Dyes as a Versatile Tool for Fluorescence Microscopy,” J. Am. Chem. Soc. 135(4), 1197–1200 (2013).
[Crossref] [PubMed]

M. Bates, G. T. Dempsey, K. H. Chen, and X. Zhuang, “Multicolor Super-Resolution Fluorescence Imaging via Multi-Parameter Fluorophore Detection,” ChemPhysChem 13(1), 99–107 (2012).
[Crossref] [PubMed]

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8(12), 1027–1036 (2011).
[Crossref] [PubMed]

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317(5845), 1749–1753 (2007).
[Crossref] [PubMed]

Dulac, C.

A. Dani, B. Huang, J. Bergan, C. Dulac, and X. Zhuang, “Superresolution Imaging of Chemical Synapses in the Brain,” Neuron 68(5), 843–856 (2010).
[Crossref] [PubMed]

Eggeling, C.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, “Multicolor Fluorescence Nanoscopy in Fixed and Living Cells by Exciting Conventional Fluorophores with a Single Wavelength,” Biophys. J. 99(8), 2686–2694 (2010).
[Crossref] [PubMed]

Eghlidi, H.

J. Ries, C. Kaplan, E. Platonova, H. Eghlidi, and H. Ewers, “A simple, versatile method for GFP-based super-resolution microscopy via nanobodies,” Nat. Methods 9(6), 582–584 (2012).
[Crossref] [PubMed]

Ewers, H.

J. Ries, C. Kaplan, E. Platonova, H. Eghlidi, and H. Ewers, “A simple, versatile method for GFP-based super-resolution microscopy via nanobodies,” Nat. Methods 9(6), 582–584 (2012).
[Crossref] [PubMed]

Fölling, J.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, “Multicolor Fluorescence Nanoscopy in Fixed and Living Cells by Exciting Conventional Fluorophores with a Single Wavelength,” Biophys. J. 99(8), 2686–2694 (2010).
[Crossref] [PubMed]

Galbraith, C. G.

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, “Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes,” Proc. Natl. Acad. Sci. U.S.A. 104(51), 20308–20313 (2007).
[Crossref] [PubMed]

Galbraith, J. A.

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, “Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes,” Proc. Natl. Acad. Sci. U.S.A. 104(51), 20308–20313 (2007).
[Crossref] [PubMed]

Gillette, J.

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, “Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes,” Proc. Natl. Acad. Sci. U.S.A. 104(51), 20308–20313 (2007).
[Crossref] [PubMed]

Girirajan, T. P. K.

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[Crossref] [PubMed]

Gönczy, P.

N. Olivier, D. Keller, P. Gönczy, and S. Manley, “Resolution Doubling In 3D-STORM Imaging through Improved Buffers,” PLoS ONE 8(7), e69004 (2013).
[Crossref] [PubMed]

Heilemann, 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]

Hell, S. W.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, “Multicolor Fluorescence Nanoscopy in Fixed and Living Cells by Exciting Conventional Fluorophores with a Single Wavelength,” Biophys. J. 99(8), 2686–2694 (2010).
[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]

Hess, S. T.

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[Crossref] [PubMed]

Horn, B. K. P.

Huang, B.

R. McGorty, D. Kamiyama, and B. Huang, “Active microscope stabilization in three dimensions using image correlation,” Opt .Nanoscopy 2(1), 3 (2013).
[Crossref] [PubMed]

A. Dani, B. Huang, J. Bergan, C. Dulac, and X. Zhuang, “Superresolution Imaging of Chemical Synapses in the Brain,” Neuron 68(5), 843–856 (2010).
[Crossref] [PubMed]

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317(5845), 1749–1753 (2007).
[Crossref] [PubMed]

Jakobs, S.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, “Multicolor Fluorescence Nanoscopy in Fixed and Living Cells by Exciting Conventional Fluorophores with a Single Wavelength,” Biophys. J. 99(8), 2686–2694 (2010).
[Crossref] [PubMed]

Jungmann, R.

R. Jungmann, M. S. Avendaño, J. B. Woehrstein, M. Dai, W. M. Shih, and P. Yin, “Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT,” Nat. Methods 11(3), 313–318 (2014).
[Crossref] [PubMed]

Kamiyama, D.

R. McGorty, D. Kamiyama, and B. Huang, “Active microscope stabilization in three dimensions using image correlation,” Opt .Nanoscopy 2(1), 3 (2013).
[Crossref] [PubMed]

Kaplan, C.

J. Ries, C. Kaplan, E. Platonova, H. Eghlidi, and H. Ewers, “A simple, versatile method for GFP-based super-resolution microscopy via nanobodies,” Nat. Methods 9(6), 582–584 (2012).
[Crossref] [PubMed]

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]

Keller, D.

N. Olivier, D. Keller, P. Gönczy, and S. Manley, “Resolution Doubling In 3D-STORM Imaging through Improved Buffers,” PLoS ONE 8(7), e69004 (2013).
[Crossref] [PubMed]

Koo, P. K.

P. K. Koo, S. U. Setru, and S. G. J. Mochrie, “Active drift stabilization in three dimensions via image cross-correlation,” Rev. Sci. Instrum. 84(10), 103705 (2013).
[Crossref] [PubMed]

Lakadamyali, M.

J. Tam, G. A. Cordier, J. S. Borbely, Á. Sandoval Álvarez, and M. Lakadamyali, “Cross-Talk-Free Multi-Color STORM Imaging Using a Single Fluorophore,” PLoS ONE 9(7), e101772 (2014).
[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]

Looger, L. L.

J. Tang, J. Akerboom, A. Vaziri, L. L. Looger, and C. V. Shank, “Near-isotropic 3D optical nanoscopy with photon-limited chromophores,” Proc. Natl. Acad. Sci. U.S.A. 107(22), 10068–10073 (2010).
[Crossref] [PubMed]

Manley, S.

N. Olivier, D. Keller, P. Gönczy, and S. Manley, “Resolution Doubling In 3D-STORM Imaging through Improved Buffers,” PLoS ONE 8(7), e69004 (2013).
[Crossref] [PubMed]

Mason, M. D.

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[Crossref] [PubMed]

McGorty, R.

R. McGorty, D. Kamiyama, and B. Huang, “Active microscope stabilization in three dimensions using image correlation,” Opt .Nanoscopy 2(1), 3 (2013).
[Crossref] [PubMed]

Medda, R.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, “Multicolor Fluorescence Nanoscopy in Fixed and Living Cells by Exciting Conventional Fluorophores with a Single Wavelength,” Biophys. J. 99(8), 2686–2694 (2010).
[Crossref] [PubMed]

Mochrie, S. G. J.

P. K. Koo, S. U. Setru, and S. G. J. Mochrie, “Active drift stabilization in three dimensions via image cross-correlation,” Rev. Sci. Instrum. 84(10), 103705 (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]

Olenych, S.

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, “Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes,” Proc. Natl. Acad. Sci. U.S.A. 104(51), 20308–20313 (2007).
[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]

Olivier, N.

N. Olivier, D. Keller, P. Gönczy, and S. Manley, “Resolution Doubling In 3D-STORM Imaging through Improved Buffers,” PLoS ONE 8(7), e69004 (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]

Platonova, E.

J. Ries, C. Kaplan, E. Platonova, H. Eghlidi, and H. Ewers, “A simple, versatile method for GFP-based super-resolution microscopy via nanobodies,” Nat. Methods 9(6), 582–584 (2012).
[Crossref] [PubMed]

Ries, J.

J. Ries, C. Kaplan, E. Platonova, H. Eghlidi, and H. Ewers, “A simple, versatile method for GFP-based super-resolution microscopy via nanobodies,” Nat. Methods 9(6), 582–584 (2012).
[Crossref] [PubMed]

Rothermel, E.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, “Multicolor Fluorescence Nanoscopy in Fixed and Living Cells by Exciting Conventional Fluorophores with a Single Wavelength,” Biophys. J. 99(8), 2686–2694 (2010).
[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]

Sandoval Álvarez, Á.

J. Tam, G. A. Cordier, J. S. Borbely, Á. Sandoval Álvarez, and M. Lakadamyali, “Cross-Talk-Free Multi-Color STORM Imaging Using a Single Fluorophore,” PLoS ONE 9(7), e101772 (2014).
[Crossref] [PubMed]

Sauer, 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]

Schönle, A.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, “Multicolor Fluorescence Nanoscopy in Fixed and Living Cells by Exciting Conventional Fluorophores with a Single Wavelength,” Biophys. J. 99(8), 2686–2694 (2010).
[Crossref] [PubMed]

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]

Setru, S. U.

P. K. Koo, S. U. Setru, and S. G. J. Mochrie, “Active drift stabilization in three dimensions via image cross-correlation,” Rev. Sci. Instrum. 84(10), 103705 (2013).
[Crossref] [PubMed]

Shank, C. V.

J. Tang, J. Akerboom, A. Vaziri, L. L. Looger, and C. V. Shank, “Near-isotropic 3D optical nanoscopy with photon-limited chromophores,” Proc. Natl. Acad. Sci. U.S.A. 107(22), 10068–10073 (2010).
[Crossref] [PubMed]

Shih, W. M.

R. Jungmann, M. S. Avendaño, J. B. Woehrstein, M. Dai, W. M. Shih, and P. Yin, “Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT,” Nat. Methods 11(3), 313–318 (2014).
[Crossref] [PubMed]

Shroff, H.

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, “Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes,” Proc. Natl. Acad. Sci. U.S.A. 104(51), 20308–20313 (2007).
[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]

Sun, E.

J. C. Vaughan, G. T. Dempsey, E. Sun, and X. Zhuang, “Phosphine Quenching of Cyanine Dyes as a Versatile Tool for Fluorescence Microscopy,” J. Am. Chem. Soc. 135(4), 1197–1200 (2013).
[Crossref] [PubMed]

Tam, J.

J. Tam, G. A. Cordier, J. S. Borbely, Á. Sandoval Álvarez, and M. Lakadamyali, “Cross-Talk-Free Multi-Color STORM Imaging Using a Single Fluorophore,” PLoS ONE 9(7), e101772 (2014).
[Crossref] [PubMed]

Tang, J.

J. Tang, J. Akerboom, A. Vaziri, L. L. Looger, and C. V. Shank, “Near-isotropic 3D optical nanoscopy with photon-limited chromophores,” Proc. Natl. Acad. Sci. U.S.A. 107(22), 10068–10073 (2010).
[Crossref] [PubMed]

Testa, I.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, “Multicolor Fluorescence Nanoscopy in Fixed and Living Cells by Exciting Conventional Fluorophores with a Single Wavelength,” Biophys. J. 99(8), 2686–2694 (2010).
[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]

van de Linde, S.

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]

Vaughan, J. C.

J. C. Vaughan, G. T. Dempsey, E. Sun, and X. Zhuang, “Phosphine Quenching of Cyanine Dyes as a Versatile Tool for Fluorescence Microscopy,” J. Am. Chem. Soc. 135(4), 1197–1200 (2013).
[Crossref] [PubMed]

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8(12), 1027–1036 (2011).
[Crossref] [PubMed]

Vaziri, A.

J. Tang, J. Akerboom, A. Vaziri, L. L. Looger, and C. V. Shank, “Near-isotropic 3D optical nanoscopy with photon-limited chromophores,” Proc. Natl. Acad. Sci. U.S.A. 107(22), 10068–10073 (2010).
[Crossref] [PubMed]

von Middendorf, C.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, “Multicolor Fluorescence Nanoscopy in Fixed and Living Cells by Exciting Conventional Fluorophores with a Single Wavelength,” Biophys. J. 99(8), 2686–2694 (2010).
[Crossref] [PubMed]

White, H.

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, “Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes,” Proc. Natl. Acad. Sci. U.S.A. 104(51), 20308–20313 (2007).
[Crossref] [PubMed]

Woehrstein, J. B.

R. Jungmann, M. S. Avendaño, J. B. Woehrstein, M. Dai, W. M. Shih, and P. Yin, “Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT,” Nat. Methods 11(3), 313–318 (2014).
[Crossref] [PubMed]

Wurm, C. A.

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, “Multicolor Fluorescence Nanoscopy in Fixed and Living Cells by Exciting Conventional Fluorophores with a Single Wavelength,” Biophys. J. 99(8), 2686–2694 (2010).
[Crossref] [PubMed]

Yin, P.

R. Jungmann, M. S. Avendaño, J. B. Woehrstein, M. Dai, W. M. Shih, and P. Yin, “Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT,” Nat. Methods 11(3), 313–318 (2014).
[Crossref] [PubMed]

Zhuang, X.

J. C. Vaughan, G. T. Dempsey, E. Sun, and X. Zhuang, “Phosphine Quenching of Cyanine Dyes as a Versatile Tool for Fluorescence Microscopy,” J. Am. Chem. Soc. 135(4), 1197–1200 (2013).
[Crossref] [PubMed]

M. Bates, G. T. Dempsey, K. H. Chen, and X. Zhuang, “Multicolor Super-Resolution Fluorescence Imaging via Multi-Parameter Fluorophore Detection,” ChemPhysChem 13(1), 99–107 (2012).
[Crossref] [PubMed]

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8(12), 1027–1036 (2011).
[Crossref] [PubMed]

A. Dani, B. Huang, J. Bergan, C. Dulac, and X. Zhuang, “Superresolution Imaging of Chemical Synapses in the Brain,” Neuron 68(5), 843–856 (2010).
[Crossref] [PubMed]

M. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317(5845), 1749–1753 (2007).
[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]

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]

Biophys. J. (2)

S. T. Hess, T. P. K. Girirajan, and M. D. Mason, “Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy,” Biophys. J. 91(11), 4258–4272 (2006).
[Crossref] [PubMed]

I. Testa, C. A. Wurm, R. Medda, E. Rothermel, C. von Middendorf, J. Fölling, S. Jakobs, A. Schönle, S. W. Hell, and C. Eggeling, “Multicolor Fluorescence Nanoscopy in Fixed and Living Cells by Exciting Conventional Fluorophores with a Single Wavelength,” Biophys. J. 99(8), 2686–2694 (2010).
[Crossref] [PubMed]

ChemPhysChem (1)

M. Bates, G. T. Dempsey, K. H. Chen, and X. Zhuang, “Multicolor Super-Resolution Fluorescence Imaging via Multi-Parameter Fluorophore Detection,” ChemPhysChem 13(1), 99–107 (2012).
[Crossref] [PubMed]

J. Am. Chem. Soc. (1)

J. C. Vaughan, G. T. Dempsey, E. Sun, and X. Zhuang, “Phosphine Quenching of Cyanine Dyes as a Versatile Tool for Fluorescence Microscopy,” J. Am. Chem. Soc. 135(4), 1197–1200 (2013).
[Crossref] [PubMed]

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

Nat. Methods (4)

J. Ries, C. Kaplan, E. Platonova, H. Eghlidi, and H. Ewers, “A simple, versatile method for GFP-based super-resolution microscopy via nanobodies,” Nat. Methods 9(6), 582–584 (2012).
[Crossref] [PubMed]

R. Jungmann, M. S. Avendaño, J. B. Woehrstein, M. Dai, W. M. Shih, and P. Yin, “Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT,” Nat. Methods 11(3), 313–318 (2014).
[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]

G. T. Dempsey, J. C. Vaughan, K. H. Chen, M. Bates, and X. Zhuang, “Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging,” Nat. Methods 8(12), 1027–1036 (2011).
[Crossref] [PubMed]

Neuron (1)

A. Dani, B. Huang, J. Bergan, C. Dulac, and X. Zhuang, “Superresolution Imaging of Chemical Synapses in the Brain,” Neuron 68(5), 843–856 (2010).
[Crossref] [PubMed]

Opt .Nanoscopy (1)

R. McGorty, D. Kamiyama, and B. Huang, “Active microscope stabilization in three dimensions using image correlation,” Opt .Nanoscopy 2(1), 3 (2013).
[Crossref] [PubMed]

PLoS ONE (2)

N. Olivier, D. Keller, P. Gönczy, and S. Manley, “Resolution Doubling In 3D-STORM Imaging through Improved Buffers,” PLoS ONE 8(7), e69004 (2013).
[Crossref] [PubMed]

J. Tam, G. A. Cordier, J. S. Borbely, Á. Sandoval Álvarez, and M. Lakadamyali, “Cross-Talk-Free Multi-Color STORM Imaging Using a Single Fluorophore,” PLoS ONE 9(7), e101772 (2014).
[Crossref] [PubMed]

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

H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M. W. Davidson, and E. Betzig, “Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes,” Proc. Natl. Acad. Sci. U.S.A. 104(51), 20308–20313 (2007).
[Crossref] [PubMed]

J. Tang, J. Akerboom, A. Vaziri, L. L. Looger, and C. V. Shank, “Near-isotropic 3D optical nanoscopy with photon-limited chromophores,” Proc. Natl. Acad. Sci. U.S.A. 107(22), 10068–10073 (2010).
[Crossref] [PubMed]

Rev. Sci. Instrum. (1)

P. K. Koo, S. U. Setru, and S. G. J. Mochrie, “Active drift stabilization in three dimensions via image cross-correlation,” Rev. Sci. Instrum. 84(10), 103705 (2013).
[Crossref] [PubMed]

Science (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. Bates, B. Huang, G. T. Dempsey, and X. Zhuang, “Multicolor Super-Resolution Imaging with Photo-Switchable Fluorescent Probes,” Science 317(5845), 1749–1753 (2007).
[Crossref] [PubMed]

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Figures (7)

Fig. 1
Fig. 1

The photon counts distribution of Alexa647 and Alexa750 in TCEP only buffer and TCEP + COT buffer. TCEP + COT buffer doubles the photon emission during photoswitching while keeping a good balance between the two dyes. Data measured under the excitation power of 4kW/cm2 for 656nm laser and 4.5kW/cm2 for 750nm laser.

Fig. 2
Fig. 2

The on-time distributions of Alexa647 and Alexa750 in optimized TCEP + COT imaging buffer show a good match between channels. Mean τon for Alexa647 and Alexa750 are 100ms and 108ms, respectively. Data measured under the excitation power of 4kW/cm2 for 656nm laser and 4.5kW/cm2 for 750nm laser.

Fig. 3
Fig. 3

The schematics of a two-color localization microscope with split-channel type detection path. DM1: multiband dichroic mirror (Chroma) that reflects excitation/activation lasers and transmits fluorescent signals. DM2: short pass dichroic mirror (Chroma) cut off at 650nm that reflects fluorescent signals and transmits the scattered image from sample. DM3 is a short pass dichroic mirror (Chroma) cut off at 755nm to split the two fluorescent channels. DM4 and DM5 are laser beam combiners (Thorlabs). M stands for mirrors. F1 and F2 are identical bandpass filters (Chroma) that block the laser and fluorescence from sample thus let locking camera only accept scattered image from sample. F3 and F4 are the emission filters (Chroma) for Alexa750 and Alexa647, respectively. The inset shows super resolution image profiles of Alexa647 and Alexa750 single molecules. The FWHM widths are under 15nm in both channels, consistent with δ = PSF/√N where N is the photon number.

Fig. 4
Fig. 4

The performance of active sample locking. NCC tracking of a 1.2μm polystyrene bead measures sample movement. The bead produces scattered imaging with SNR>10, allowing tracking with sub nanometer resolution. Insertion zooms in the marked area showing the locking is able to stabilize sample with nanometer resolution.

Fig. 5
Fig. 5

Two-color super resolution imaging resolves p32 (mitochondrial matrix) packed by Tom20 (mitochondrial outer membrane) in HEK293 cell line. (a) Epi fluorescent image with totally blurred structure. (b) The super resolution images at the same location. (c) The transvers profile correspondence to boxed region in (b), showing clear the p32 is packed inside mitochondrial outer membrane. Scale bars: 1μm.

Fig. 6
Fig. 6

Synaptic structure in tissue slice resolved by localization microscope. (a)(c) and (e) show the EPI-fluorescent image which fails to tell the details of structure. (b)(d) and (f) are superresolution images acquired by designed two-color localization microscope. The pre- and post-synaptic structures are clearly resolved. Scale bars: 1μm in (a) and (b); 200nm in (c) and (d).

Fig. 7
Fig. 7

Two-color super resolution imaging of Alexa750-anti-GFP nanobody labeled Tub1 of spindle microtubule and Alexa647-anti-RFP nanobody labeled Tub4 of spindle pole body in a yeast cell. (a) and (b) are Epi fluorescent images where (b) is the zoom in of the marked area in (a). (c) and (d) are the two-color super resolution images at the same position where the anchoring of spindle microtubules to spindle pole bodies can be clearly resolved. Scale bar: 1μm in (a) and (b); 200nm in (c) and (d).

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