Abstract

Multicolour single molecule fluorescence imaging enables the study of multiple proteins in the membranes of living cells. We describe the use of a supercontinuum laser as the excitation source, show its comparability with multiplexed single-wavelength lasers and demonstrate that it can be used to study membrane proteins such as the ErbB receptor family. We discuss the benefits of white-light sources for single molecule fluorescence, in particular their ease of use and the freedom to use the most appropriate dye without being constrained by available laser wavelengths.

© 2012 OSA

Full Article  |  PDF Article

References

  • View by:
  • |
  • |
  • |

  1. Y. Yarden and M. X. Sliwkowski, “Untangling the ErbB signalling network,” Nat. Rev. Mol. Cell Biol.2(2), 127–137 (2001).
    [CrossRef] [PubMed]
  2. P. R. Selvin and T. Ha, Single Molecule Techniques: a Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, 2007).
  3. Y. Sako, “Imaging single molecules in living cells for systems biology,” Mol. Syst. Biol.2, 56 (2006).
    [CrossRef] [PubMed]
  4. D. Axelrod, “Total internal reflection fluorescence microscopy in cell biology,” Traffic2(11), 764–774 (2001).
    [CrossRef] [PubMed]
  5. J. A. Hern, A. H. Baig, G. I. Mashanov, B. Birdsall, J. E. T. Corrie, S. Lazareno, J. E. Molloy, and N. J. M. Birdsall, “Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules,” Proc. Natl. Acad. Sci. U.S.A.107(6), 2693–2698 (2010).
    [CrossRef] [PubMed]
  6. K. Ritchie, X. Y. Shan, J. Kondo, K. Iwasawa, T. Fujiwara, and A. Kusumi, “Detection of non-Brownian diffusion in the cell membrane in single molecule tracking,” Biophys. J.88(3), 2266–2277 (2005).
    [CrossRef] [PubMed]
  7. P. D. Dunne, R. A. Fernandes, J. McColl, J. W. Yoon, J. R. James, S. J. Davis, and D. Klenerman, “DySCo: quantitating associations of membrane proteins using two-color single-molecule tracking,” Biophys. J.97(4), L5–L7 (2009).
    [CrossRef] [PubMed]
  8. J. F. Beausang, H. W. Schroeder, P. C. Nelson, and Y. E. Goldman, “Twirling of actin by myosins II and V observed via polarized TIRF in a modified gliding assay,” Biophys. J.95(12), 5820–5831 (2008).
    [CrossRef] [PubMed]
  9. V. C. DeRocco, T. Anderson, J. Piehler, D. A. Erie, and K. Weninger, “Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes,” Biotechniques49(5), 807–816 (2010).
    [CrossRef] [PubMed]
  10. J. Vogelsang, T. Cordes, C. Forthmann, C. Steinhauer, and P. Tinnefeld, “Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy,” Proc. Natl. Acad. Sci. U.S.A.106(20), 8107–8112 (2009).
    [CrossRef] [PubMed]
  11. D. M. Owen, E. Auksorius, H. B. Manning, C. B. Talbot, P. A. A. de Beule, C. Dunsby, M. A. A. Neil, and P. M. W. French, “Excitation-resolved hyperspectral fluorescence lifetime imaging using a UV-extended supercontinuum source,” Opt. Lett.32(23), 3408–3410 (2007).
    [CrossRef] [PubMed]
  12. D. Wildanger, E. Rittweger, L. Kastrup, and S. W. Hell, “STED microscopy with a supercontinuum laser source,” Opt. Express16(13), 9614–9621 (2008).
    [CrossRef] [PubMed]
  13. Y. Sako, S. Minoghchi, and T. Yanagida, “Single-molecule imaging of EGFR signalling on the surface of living cells,” Nat. Cell Biol.2(3), 168–172 (2000).
    [CrossRef] [PubMed]
  14. D. J. Rolfe, C. I. McLachlan, M. Hirsch, S. R. Needham, C. J. Tynan, S. E. D. Webb, M. L. Martin-Fernandez, and M. P. Hobson, “Automated multidimensional single molecule fluorescence microscopy feature detection and tracking,” Eur. Biophys. J.40(10), 1167–1186 (2011).
    [CrossRef] [PubMed]
  15. P. Blandin, S. Lévêque-Fort, S. Lécart, J. C. Cossec, M. C. Potier, Z. Lenkei, F. Druon, and P. Georges, “Time-gated total internal reflection fluorescence microscopy with a supercontinuum excitation source,” Appl. Opt.48(3), 553–559 (2009).
    [CrossRef] [PubMed]
  16. B. Agnarsson, S. Ingthorsson, T. Gudjonsson, and K. Leosson, “Evanescent-wave fluorescence microscopy using symmetric planar waveguides,” Opt. Express17(7), 5075–5082 (2009).
    [CrossRef] [PubMed]
  17. J. P. Knemeyer, D. P. Herten, and M. Sauer, “Detection and identification of single molecules in living cells using spectrally resolved fluorescence lifetime imaging microscopy,” Anal. Chem.75(9), 2147–2153 (2003).
    [CrossRef] [PubMed]

2011 (1)

D. J. Rolfe, C. I. McLachlan, M. Hirsch, S. R. Needham, C. J. Tynan, S. E. D. Webb, M. L. Martin-Fernandez, and M. P. Hobson, “Automated multidimensional single molecule fluorescence microscopy feature detection and tracking,” Eur. Biophys. J.40(10), 1167–1186 (2011).
[CrossRef] [PubMed]

2010 (2)

J. A. Hern, A. H. Baig, G. I. Mashanov, B. Birdsall, J. E. T. Corrie, S. Lazareno, J. E. Molloy, and N. J. M. Birdsall, “Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules,” Proc. Natl. Acad. Sci. U.S.A.107(6), 2693–2698 (2010).
[CrossRef] [PubMed]

V. C. DeRocco, T. Anderson, J. Piehler, D. A. Erie, and K. Weninger, “Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes,” Biotechniques49(5), 807–816 (2010).
[CrossRef] [PubMed]

2009 (4)

J. Vogelsang, T. Cordes, C. Forthmann, C. Steinhauer, and P. Tinnefeld, “Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy,” Proc. Natl. Acad. Sci. U.S.A.106(20), 8107–8112 (2009).
[CrossRef] [PubMed]

P. D. Dunne, R. A. Fernandes, J. McColl, J. W. Yoon, J. R. James, S. J. Davis, and D. Klenerman, “DySCo: quantitating associations of membrane proteins using two-color single-molecule tracking,” Biophys. J.97(4), L5–L7 (2009).
[CrossRef] [PubMed]

P. Blandin, S. Lévêque-Fort, S. Lécart, J. C. Cossec, M. C. Potier, Z. Lenkei, F. Druon, and P. Georges, “Time-gated total internal reflection fluorescence microscopy with a supercontinuum excitation source,” Appl. Opt.48(3), 553–559 (2009).
[CrossRef] [PubMed]

B. Agnarsson, S. Ingthorsson, T. Gudjonsson, and K. Leosson, “Evanescent-wave fluorescence microscopy using symmetric planar waveguides,” Opt. Express17(7), 5075–5082 (2009).
[CrossRef] [PubMed]

2008 (2)

D. Wildanger, E. Rittweger, L. Kastrup, and S. W. Hell, “STED microscopy with a supercontinuum laser source,” Opt. Express16(13), 9614–9621 (2008).
[CrossRef] [PubMed]

J. F. Beausang, H. W. Schroeder, P. C. Nelson, and Y. E. Goldman, “Twirling of actin by myosins II and V observed via polarized TIRF in a modified gliding assay,” Biophys. J.95(12), 5820–5831 (2008).
[CrossRef] [PubMed]

2007 (1)

2006 (1)

Y. Sako, “Imaging single molecules in living cells for systems biology,” Mol. Syst. Biol.2, 56 (2006).
[CrossRef] [PubMed]

2005 (1)

K. Ritchie, X. Y. Shan, J. Kondo, K. Iwasawa, T. Fujiwara, and A. Kusumi, “Detection of non-Brownian diffusion in the cell membrane in single molecule tracking,” Biophys. J.88(3), 2266–2277 (2005).
[CrossRef] [PubMed]

2003 (1)

J. P. Knemeyer, D. P. Herten, and M. Sauer, “Detection and identification of single molecules in living cells using spectrally resolved fluorescence lifetime imaging microscopy,” Anal. Chem.75(9), 2147–2153 (2003).
[CrossRef] [PubMed]

2001 (2)

Y. Yarden and M. X. Sliwkowski, “Untangling the ErbB signalling network,” Nat. Rev. Mol. Cell Biol.2(2), 127–137 (2001).
[CrossRef] [PubMed]

D. Axelrod, “Total internal reflection fluorescence microscopy in cell biology,” Traffic2(11), 764–774 (2001).
[CrossRef] [PubMed]

2000 (1)

Y. Sako, S. Minoghchi, and T. Yanagida, “Single-molecule imaging of EGFR signalling on the surface of living cells,” Nat. Cell Biol.2(3), 168–172 (2000).
[CrossRef] [PubMed]

Agnarsson, B.

Anderson, T.

V. C. DeRocco, T. Anderson, J. Piehler, D. A. Erie, and K. Weninger, “Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes,” Biotechniques49(5), 807–816 (2010).
[CrossRef] [PubMed]

Auksorius, E.

Axelrod, D.

D. Axelrod, “Total internal reflection fluorescence microscopy in cell biology,” Traffic2(11), 764–774 (2001).
[CrossRef] [PubMed]

Baig, A. H.

J. A. Hern, A. H. Baig, G. I. Mashanov, B. Birdsall, J. E. T. Corrie, S. Lazareno, J. E. Molloy, and N. J. M. Birdsall, “Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules,” Proc. Natl. Acad. Sci. U.S.A.107(6), 2693–2698 (2010).
[CrossRef] [PubMed]

Beausang, J. F.

J. F. Beausang, H. W. Schroeder, P. C. Nelson, and Y. E. Goldman, “Twirling of actin by myosins II and V observed via polarized TIRF in a modified gliding assay,” Biophys. J.95(12), 5820–5831 (2008).
[CrossRef] [PubMed]

Birdsall, B.

J. A. Hern, A. H. Baig, G. I. Mashanov, B. Birdsall, J. E. T. Corrie, S. Lazareno, J. E. Molloy, and N. J. M. Birdsall, “Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules,” Proc. Natl. Acad. Sci. U.S.A.107(6), 2693–2698 (2010).
[CrossRef] [PubMed]

Birdsall, N. J. M.

J. A. Hern, A. H. Baig, G. I. Mashanov, B. Birdsall, J. E. T. Corrie, S. Lazareno, J. E. Molloy, and N. J. M. Birdsall, “Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules,” Proc. Natl. Acad. Sci. U.S.A.107(6), 2693–2698 (2010).
[CrossRef] [PubMed]

Blandin, P.

Cordes, T.

J. Vogelsang, T. Cordes, C. Forthmann, C. Steinhauer, and P. Tinnefeld, “Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy,” Proc. Natl. Acad. Sci. U.S.A.106(20), 8107–8112 (2009).
[CrossRef] [PubMed]

Corrie, J. E. T.

J. A. Hern, A. H. Baig, G. I. Mashanov, B. Birdsall, J. E. T. Corrie, S. Lazareno, J. E. Molloy, and N. J. M. Birdsall, “Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules,” Proc. Natl. Acad. Sci. U.S.A.107(6), 2693–2698 (2010).
[CrossRef] [PubMed]

Cossec, J. C.

Davis, S. J.

P. D. Dunne, R. A. Fernandes, J. McColl, J. W. Yoon, J. R. James, S. J. Davis, and D. Klenerman, “DySCo: quantitating associations of membrane proteins using two-color single-molecule tracking,” Biophys. J.97(4), L5–L7 (2009).
[CrossRef] [PubMed]

de Beule, P. A. A.

DeRocco, V. C.

V. C. DeRocco, T. Anderson, J. Piehler, D. A. Erie, and K. Weninger, “Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes,” Biotechniques49(5), 807–816 (2010).
[CrossRef] [PubMed]

Druon, F.

Dunne, P. D.

P. D. Dunne, R. A. Fernandes, J. McColl, J. W. Yoon, J. R. James, S. J. Davis, and D. Klenerman, “DySCo: quantitating associations of membrane proteins using two-color single-molecule tracking,” Biophys. J.97(4), L5–L7 (2009).
[CrossRef] [PubMed]

Dunsby, C.

Erie, D. A.

V. C. DeRocco, T. Anderson, J. Piehler, D. A. Erie, and K. Weninger, “Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes,” Biotechniques49(5), 807–816 (2010).
[CrossRef] [PubMed]

Fernandes, R. A.

P. D. Dunne, R. A. Fernandes, J. McColl, J. W. Yoon, J. R. James, S. J. Davis, and D. Klenerman, “DySCo: quantitating associations of membrane proteins using two-color single-molecule tracking,” Biophys. J.97(4), L5–L7 (2009).
[CrossRef] [PubMed]

Forthmann, C.

J. Vogelsang, T. Cordes, C. Forthmann, C. Steinhauer, and P. Tinnefeld, “Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy,” Proc. Natl. Acad. Sci. U.S.A.106(20), 8107–8112 (2009).
[CrossRef] [PubMed]

French, P. M. W.

Fujiwara, T.

K. Ritchie, X. Y. Shan, J. Kondo, K. Iwasawa, T. Fujiwara, and A. Kusumi, “Detection of non-Brownian diffusion in the cell membrane in single molecule tracking,” Biophys. J.88(3), 2266–2277 (2005).
[CrossRef] [PubMed]

Georges, P.

Goldman, Y. E.

J. F. Beausang, H. W. Schroeder, P. C. Nelson, and Y. E. Goldman, “Twirling of actin by myosins II and V observed via polarized TIRF in a modified gliding assay,” Biophys. J.95(12), 5820–5831 (2008).
[CrossRef] [PubMed]

Gudjonsson, T.

Hell, S. W.

Hern, J. A.

J. A. Hern, A. H. Baig, G. I. Mashanov, B. Birdsall, J. E. T. Corrie, S. Lazareno, J. E. Molloy, and N. J. M. Birdsall, “Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules,” Proc. Natl. Acad. Sci. U.S.A.107(6), 2693–2698 (2010).
[CrossRef] [PubMed]

Herten, D. P.

J. P. Knemeyer, D. P. Herten, and M. Sauer, “Detection and identification of single molecules in living cells using spectrally resolved fluorescence lifetime imaging microscopy,” Anal. Chem.75(9), 2147–2153 (2003).
[CrossRef] [PubMed]

Hirsch, M.

D. J. Rolfe, C. I. McLachlan, M. Hirsch, S. R. Needham, C. J. Tynan, S. E. D. Webb, M. L. Martin-Fernandez, and M. P. Hobson, “Automated multidimensional single molecule fluorescence microscopy feature detection and tracking,” Eur. Biophys. J.40(10), 1167–1186 (2011).
[CrossRef] [PubMed]

Hobson, M. P.

D. J. Rolfe, C. I. McLachlan, M. Hirsch, S. R. Needham, C. J. Tynan, S. E. D. Webb, M. L. Martin-Fernandez, and M. P. Hobson, “Automated multidimensional single molecule fluorescence microscopy feature detection and tracking,” Eur. Biophys. J.40(10), 1167–1186 (2011).
[CrossRef] [PubMed]

Ingthorsson, S.

Iwasawa, K.

K. Ritchie, X. Y. Shan, J. Kondo, K. Iwasawa, T. Fujiwara, and A. Kusumi, “Detection of non-Brownian diffusion in the cell membrane in single molecule tracking,” Biophys. J.88(3), 2266–2277 (2005).
[CrossRef] [PubMed]

James, J. R.

P. D. Dunne, R. A. Fernandes, J. McColl, J. W. Yoon, J. R. James, S. J. Davis, and D. Klenerman, “DySCo: quantitating associations of membrane proteins using two-color single-molecule tracking,” Biophys. J.97(4), L5–L7 (2009).
[CrossRef] [PubMed]

Kastrup, L.

Klenerman, D.

P. D. Dunne, R. A. Fernandes, J. McColl, J. W. Yoon, J. R. James, S. J. Davis, and D. Klenerman, “DySCo: quantitating associations of membrane proteins using two-color single-molecule tracking,” Biophys. J.97(4), L5–L7 (2009).
[CrossRef] [PubMed]

Knemeyer, J. P.

J. P. Knemeyer, D. P. Herten, and M. Sauer, “Detection and identification of single molecules in living cells using spectrally resolved fluorescence lifetime imaging microscopy,” Anal. Chem.75(9), 2147–2153 (2003).
[CrossRef] [PubMed]

Kondo, J.

K. Ritchie, X. Y. Shan, J. Kondo, K. Iwasawa, T. Fujiwara, and A. Kusumi, “Detection of non-Brownian diffusion in the cell membrane in single molecule tracking,” Biophys. J.88(3), 2266–2277 (2005).
[CrossRef] [PubMed]

Kusumi, A.

K. Ritchie, X. Y. Shan, J. Kondo, K. Iwasawa, T. Fujiwara, and A. Kusumi, “Detection of non-Brownian diffusion in the cell membrane in single molecule tracking,” Biophys. J.88(3), 2266–2277 (2005).
[CrossRef] [PubMed]

Lazareno, S.

J. A. Hern, A. H. Baig, G. I. Mashanov, B. Birdsall, J. E. T. Corrie, S. Lazareno, J. E. Molloy, and N. J. M. Birdsall, “Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules,” Proc. Natl. Acad. Sci. U.S.A.107(6), 2693–2698 (2010).
[CrossRef] [PubMed]

Lécart, S.

Lenkei, Z.

Leosson, K.

Lévêque-Fort, S.

Manning, H. B.

Martin-Fernandez, M. L.

D. J. Rolfe, C. I. McLachlan, M. Hirsch, S. R. Needham, C. J. Tynan, S. E. D. Webb, M. L. Martin-Fernandez, and M. P. Hobson, “Automated multidimensional single molecule fluorescence microscopy feature detection and tracking,” Eur. Biophys. J.40(10), 1167–1186 (2011).
[CrossRef] [PubMed]

Mashanov, G. I.

J. A. Hern, A. H. Baig, G. I. Mashanov, B. Birdsall, J. E. T. Corrie, S. Lazareno, J. E. Molloy, and N. J. M. Birdsall, “Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules,” Proc. Natl. Acad. Sci. U.S.A.107(6), 2693–2698 (2010).
[CrossRef] [PubMed]

McColl, J.

P. D. Dunne, R. A. Fernandes, J. McColl, J. W. Yoon, J. R. James, S. J. Davis, and D. Klenerman, “DySCo: quantitating associations of membrane proteins using two-color single-molecule tracking,” Biophys. J.97(4), L5–L7 (2009).
[CrossRef] [PubMed]

McLachlan, C. I.

D. J. Rolfe, C. I. McLachlan, M. Hirsch, S. R. Needham, C. J. Tynan, S. E. D. Webb, M. L. Martin-Fernandez, and M. P. Hobson, “Automated multidimensional single molecule fluorescence microscopy feature detection and tracking,” Eur. Biophys. J.40(10), 1167–1186 (2011).
[CrossRef] [PubMed]

Minoghchi, S.

Y. Sako, S. Minoghchi, and T. Yanagida, “Single-molecule imaging of EGFR signalling on the surface of living cells,” Nat. Cell Biol.2(3), 168–172 (2000).
[CrossRef] [PubMed]

Molloy, J. E.

J. A. Hern, A. H. Baig, G. I. Mashanov, B. Birdsall, J. E. T. Corrie, S. Lazareno, J. E. Molloy, and N. J. M. Birdsall, “Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules,” Proc. Natl. Acad. Sci. U.S.A.107(6), 2693–2698 (2010).
[CrossRef] [PubMed]

Needham, S. R.

D. J. Rolfe, C. I. McLachlan, M. Hirsch, S. R. Needham, C. J. Tynan, S. E. D. Webb, M. L. Martin-Fernandez, and M. P. Hobson, “Automated multidimensional single molecule fluorescence microscopy feature detection and tracking,” Eur. Biophys. J.40(10), 1167–1186 (2011).
[CrossRef] [PubMed]

Neil, M. A. A.

Nelson, P. C.

J. F. Beausang, H. W. Schroeder, P. C. Nelson, and Y. E. Goldman, “Twirling of actin by myosins II and V observed via polarized TIRF in a modified gliding assay,” Biophys. J.95(12), 5820–5831 (2008).
[CrossRef] [PubMed]

Owen, D. M.

Piehler, J.

V. C. DeRocco, T. Anderson, J. Piehler, D. A. Erie, and K. Weninger, “Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes,” Biotechniques49(5), 807–816 (2010).
[CrossRef] [PubMed]

Potier, M. C.

Ritchie, K.

K. Ritchie, X. Y. Shan, J. Kondo, K. Iwasawa, T. Fujiwara, and A. Kusumi, “Detection of non-Brownian diffusion in the cell membrane in single molecule tracking,” Biophys. J.88(3), 2266–2277 (2005).
[CrossRef] [PubMed]

Rittweger, E.

Rolfe, D. J.

D. J. Rolfe, C. I. McLachlan, M. Hirsch, S. R. Needham, C. J. Tynan, S. E. D. Webb, M. L. Martin-Fernandez, and M. P. Hobson, “Automated multidimensional single molecule fluorescence microscopy feature detection and tracking,” Eur. Biophys. J.40(10), 1167–1186 (2011).
[CrossRef] [PubMed]

Sako, Y.

Y. Sako, “Imaging single molecules in living cells for systems biology,” Mol. Syst. Biol.2, 56 (2006).
[CrossRef] [PubMed]

Y. Sako, S. Minoghchi, and T. Yanagida, “Single-molecule imaging of EGFR signalling on the surface of living cells,” Nat. Cell Biol.2(3), 168–172 (2000).
[CrossRef] [PubMed]

Sauer, M.

J. P. Knemeyer, D. P. Herten, and M. Sauer, “Detection and identification of single molecules in living cells using spectrally resolved fluorescence lifetime imaging microscopy,” Anal. Chem.75(9), 2147–2153 (2003).
[CrossRef] [PubMed]

Schroeder, H. W.

J. F. Beausang, H. W. Schroeder, P. C. Nelson, and Y. E. Goldman, “Twirling of actin by myosins II and V observed via polarized TIRF in a modified gliding assay,” Biophys. J.95(12), 5820–5831 (2008).
[CrossRef] [PubMed]

Shan, X. Y.

K. Ritchie, X. Y. Shan, J. Kondo, K. Iwasawa, T. Fujiwara, and A. Kusumi, “Detection of non-Brownian diffusion in the cell membrane in single molecule tracking,” Biophys. J.88(3), 2266–2277 (2005).
[CrossRef] [PubMed]

Sliwkowski, M. X.

Y. Yarden and M. X. Sliwkowski, “Untangling the ErbB signalling network,” Nat. Rev. Mol. Cell Biol.2(2), 127–137 (2001).
[CrossRef] [PubMed]

Steinhauer, C.

J. Vogelsang, T. Cordes, C. Forthmann, C. Steinhauer, and P. Tinnefeld, “Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy,” Proc. Natl. Acad. Sci. U.S.A.106(20), 8107–8112 (2009).
[CrossRef] [PubMed]

Talbot, C. B.

Tinnefeld, P.

J. Vogelsang, T. Cordes, C. Forthmann, C. Steinhauer, and P. Tinnefeld, “Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy,” Proc. Natl. Acad. Sci. U.S.A.106(20), 8107–8112 (2009).
[CrossRef] [PubMed]

Tynan, C. J.

D. J. Rolfe, C. I. McLachlan, M. Hirsch, S. R. Needham, C. J. Tynan, S. E. D. Webb, M. L. Martin-Fernandez, and M. P. Hobson, “Automated multidimensional single molecule fluorescence microscopy feature detection and tracking,” Eur. Biophys. J.40(10), 1167–1186 (2011).
[CrossRef] [PubMed]

Vogelsang, J.

J. Vogelsang, T. Cordes, C. Forthmann, C. Steinhauer, and P. Tinnefeld, “Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy,” Proc. Natl. Acad. Sci. U.S.A.106(20), 8107–8112 (2009).
[CrossRef] [PubMed]

Webb, S. E. D.

D. J. Rolfe, C. I. McLachlan, M. Hirsch, S. R. Needham, C. J. Tynan, S. E. D. Webb, M. L. Martin-Fernandez, and M. P. Hobson, “Automated multidimensional single molecule fluorescence microscopy feature detection and tracking,” Eur. Biophys. J.40(10), 1167–1186 (2011).
[CrossRef] [PubMed]

Weninger, K.

V. C. DeRocco, T. Anderson, J. Piehler, D. A. Erie, and K. Weninger, “Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes,” Biotechniques49(5), 807–816 (2010).
[CrossRef] [PubMed]

Wildanger, D.

Yanagida, T.

Y. Sako, S. Minoghchi, and T. Yanagida, “Single-molecule imaging of EGFR signalling on the surface of living cells,” Nat. Cell Biol.2(3), 168–172 (2000).
[CrossRef] [PubMed]

Yarden, Y.

Y. Yarden and M. X. Sliwkowski, “Untangling the ErbB signalling network,” Nat. Rev. Mol. Cell Biol.2(2), 127–137 (2001).
[CrossRef] [PubMed]

Yoon, J. W.

P. D. Dunne, R. A. Fernandes, J. McColl, J. W. Yoon, J. R. James, S. J. Davis, and D. Klenerman, “DySCo: quantitating associations of membrane proteins using two-color single-molecule tracking,” Biophys. J.97(4), L5–L7 (2009).
[CrossRef] [PubMed]

Anal. Chem. (1)

J. P. Knemeyer, D. P. Herten, and M. Sauer, “Detection and identification of single molecules in living cells using spectrally resolved fluorescence lifetime imaging microscopy,” Anal. Chem.75(9), 2147–2153 (2003).
[CrossRef] [PubMed]

Appl. Opt. (1)

Biophys. J. (3)

K. Ritchie, X. Y. Shan, J. Kondo, K. Iwasawa, T. Fujiwara, and A. Kusumi, “Detection of non-Brownian diffusion in the cell membrane in single molecule tracking,” Biophys. J.88(3), 2266–2277 (2005).
[CrossRef] [PubMed]

P. D. Dunne, R. A. Fernandes, J. McColl, J. W. Yoon, J. R. James, S. J. Davis, and D. Klenerman, “DySCo: quantitating associations of membrane proteins using two-color single-molecule tracking,” Biophys. J.97(4), L5–L7 (2009).
[CrossRef] [PubMed]

J. F. Beausang, H. W. Schroeder, P. C. Nelson, and Y. E. Goldman, “Twirling of actin by myosins II and V observed via polarized TIRF in a modified gliding assay,” Biophys. J.95(12), 5820–5831 (2008).
[CrossRef] [PubMed]

Biotechniques (1)

V. C. DeRocco, T. Anderson, J. Piehler, D. A. Erie, and K. Weninger, “Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes,” Biotechniques49(5), 807–816 (2010).
[CrossRef] [PubMed]

Eur. Biophys. J. (1)

D. J. Rolfe, C. I. McLachlan, M. Hirsch, S. R. Needham, C. J. Tynan, S. E. D. Webb, M. L. Martin-Fernandez, and M. P. Hobson, “Automated multidimensional single molecule fluorescence microscopy feature detection and tracking,” Eur. Biophys. J.40(10), 1167–1186 (2011).
[CrossRef] [PubMed]

Mol. Syst. Biol. (1)

Y. Sako, “Imaging single molecules in living cells for systems biology,” Mol. Syst. Biol.2, 56 (2006).
[CrossRef] [PubMed]

Nat. Cell Biol. (1)

Y. Sako, S. Minoghchi, and T. Yanagida, “Single-molecule imaging of EGFR signalling on the surface of living cells,” Nat. Cell Biol.2(3), 168–172 (2000).
[CrossRef] [PubMed]

Nat. Rev. Mol. Cell Biol. (1)

Y. Yarden and M. X. Sliwkowski, “Untangling the ErbB signalling network,” Nat. Rev. Mol. Cell Biol.2(2), 127–137 (2001).
[CrossRef] [PubMed]

Opt. Express (2)

Opt. Lett. (1)

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

J. A. Hern, A. H. Baig, G. I. Mashanov, B. Birdsall, J. E. T. Corrie, S. Lazareno, J. E. Molloy, and N. J. M. Birdsall, “Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules,” Proc. Natl. Acad. Sci. U.S.A.107(6), 2693–2698 (2010).
[CrossRef] [PubMed]

J. Vogelsang, T. Cordes, C. Forthmann, C. Steinhauer, and P. Tinnefeld, “Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy,” Proc. Natl. Acad. Sci. U.S.A.106(20), 8107–8112 (2009).
[CrossRef] [PubMed]

Traffic (1)

D. Axelrod, “Total internal reflection fluorescence microscopy in cell biology,” Traffic2(11), 764–774 (2001).
[CrossRef] [PubMed]

Other (1)

P. R. Selvin and T. Ha, Single Molecule Techniques: a Laboratory Manual (Cold Spring Harbor Laboratory Press, New York, 2007).

Cited By

OSA participates in CrossRef's Cited-By Linking service. Citing articles from OSA journals and other participating publishers are listed here.

Alert me when this article is cited.


Figures (5)

Fig. 1
Fig. 1

Spectra of laser sources used for multicolour imaging of Alexa 488, Alexa 546 and Atto 647N. (a) Supercontinuum with AOTF, (b) Single-wavelength lasers.

Fig. 2
Fig. 2

Multicolour total internal reflection microscope, with either a supercontinuum laser or multiplexed single-wavelength lasers as the excitation source.

Fig. 3
Fig. 3

Multicolour single molecule imaging with supercontinuum excitation. (a) White light transmission and single molecule fluorescence images of live T47D cells labelled with EGF-Alexa 488, EGF-Alexa 546 and EGF-Atto 647N, focused at the basolateral membrane. Images were acquired at 10 Hz. Scale bar = 5 µm. (Note: Image registration was performed on the acquired sub-images, which were then remapped to a common reference space to produce the images shown here.) The green squares indicate the molecules analysed in (b), whose locations within the cell are indicated by the red squares. (b) Detail images of the areas around the three analysed molecules in each wavelength band and plots of the intensities of the molecules over time. Scale bar = 1 µm.

Fig. 4
Fig. 4

Multicolour single molecule imaging with multiplexed single-wavelength laser excitation. (a) Detail images of the area around two colocalised EGF-Alexa 546 molecules on live T47D cells, in each wavelength band. Images were acquired at 10 Hz. Scale bar = 1 µm. (b) Intensities of typical EGF-Alexa 488, EGF-Alexa 546 and EGF-Atto 647N molecules.

Fig. 5
Fig. 5

Multicolour single molecule colocalisation imaging with supercontinuum laser excitation. Intensities v time of colocalised ErbB1-EGFP and EGF-Cy3.5 molecules on live CHO cells and detail images of the area around them, in each wavelength band, at the times indicated. The highlighting squares are green at time points when fluorescence features are detected and yellow when they are not. Images were acquired at 10 Hz. Scale bar = 1 µm.

Metrics