Abstract

We have developed a wide-field total-internal-reflection fluorescence microscope capable of imaging single molecules in live cells, resolved in both wavelength and polarization. We show fluorescence resonance energy transfer between single pairs of fluorescent molecules bound to signaling receptors in the plasma membrane of live cells and demonstrate the importance of polarization discrimination in addition to wavelength separation.

© 2006 Optical Society of America

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2005

J. N. Forkey, M. E. Quinlan, and Y. E. Goldman, Biophys. J. 89, 1261 (2005).
[CrossRef] [PubMed]

2004

A. L. Mattheyses, A. D. Hoppe, and D. Axelrod, Biophys. J. 87, 2787 (2004).
[CrossRef] [PubMed]

2003

S. Wakelin and C. R. Bagshaw, J. Microsc. 209, 143 (2003).
[CrossRef] [PubMed]

2002

M. Martin-Fernandez, D. T. Clarke, M. J. Tobin, S. V. Jones, and G. R. Jones, Biophys. J. 82, 2415 (2002).
[CrossRef] [PubMed]

J. Malicka, I. Gryczynski, J. Y. Fang, J. Kusba, and J. R. Lakowicz, J. Fluoresc. 12, 439 (2002).
[CrossRef]

2001

2000

L. Cognet, G. S. Harms, G. A. Blab, P. H. M. Lommerse, and T. Schmidt, Appl. Phys. Lett. 77, 4052 (2000).
[CrossRef]

Y. Sako, S. Minoghchi, and T. Yanagida, Nat. Cell Biol. 2, 168 (2000).
[CrossRef] [PubMed]

1989

D. Axelrod, Methods Cell Biol. 30, 245 (1989).
[CrossRef] [PubMed]

1984

A. R. Rees, M. Gregoriou, P. Johnson, and P. B. Garland, EMBO J. 3, 1843 (1984).
[PubMed]

1967

L. Stryer and R. P. Haugland, Proc. Natl. Acad. Sci. U.S.A. 58, 720 (1967).
[CrossRef]

Axelrod, D.

A. L. Mattheyses, A. D. Hoppe, and D. Axelrod, Biophys. J. 87, 2787 (2004).
[CrossRef] [PubMed]

D. Axelrod, Methods Cell Biol. 30, 245 (1989).
[CrossRef] [PubMed]

Bagshaw, C. R.

S. Wakelin and C. R. Bagshaw, J. Microsc. 209, 143 (2003).
[CrossRef] [PubMed]

Blab, G. A.

L. Cognet, G. S. Harms, G. A. Blab, P. H. M. Lommerse, and T. Schmidt, Appl. Phys. Lett. 77, 4052 (2000).
[CrossRef]

Clarke, D. T.

M. Martin-Fernandez, D. T. Clarke, M. J. Tobin, S. V. Jones, and G. R. Jones, Biophys. J. 82, 2415 (2002).
[CrossRef] [PubMed]

Cognet, L.

L. Cognet, G. S. Harms, G. A. Blab, P. H. M. Lommerse, and T. Schmidt, Appl. Phys. Lett. 77, 4052 (2000).
[CrossRef]

Cole, M. J.

Elson, D. S.

Fang, J. Y.

J. Malicka, I. Gryczynski, J. Y. Fang, J. Kusba, and J. R. Lakowicz, J. Fluoresc. 12, 439 (2002).
[CrossRef]

Forkey, J. N.

J. N. Forkey, M. E. Quinlan, and Y. E. Goldman, Biophys. J. 89, 1261 (2005).
[CrossRef] [PubMed]

French, P. M. W.

Garland, P. B.

A. R. Rees, M. Gregoriou, P. Johnson, and P. B. Garland, EMBO J. 3, 1843 (1984).
[PubMed]

Goldman, Y. E.

J. N. Forkey, M. E. Quinlan, and Y. E. Goldman, Biophys. J. 89, 1261 (2005).
[CrossRef] [PubMed]

Gregoriou, M.

A. R. Rees, M. Gregoriou, P. Johnson, and P. B. Garland, EMBO J. 3, 1843 (1984).
[PubMed]

Gryczynski, I.

J. Malicka, I. Gryczynski, J. Y. Fang, J. Kusba, and J. R. Lakowicz, J. Fluoresc. 12, 439 (2002).
[CrossRef]

Ha, T.

T. Ha, Methods 25, 78 (2001).
[CrossRef] [PubMed]

Harms, G. S.

L. Cognet, G. S. Harms, G. A. Blab, P. H. M. Lommerse, and T. Schmidt, Appl. Phys. Lett. 77, 4052 (2000).
[CrossRef]

Haugland, R. P.

L. Stryer and R. P. Haugland, Proc. Natl. Acad. Sci. U.S.A. 58, 720 (1967).
[CrossRef]

Hoppe, A. D.

A. L. Mattheyses, A. D. Hoppe, and D. Axelrod, Biophys. J. 87, 2787 (2004).
[CrossRef] [PubMed]

Johnson, P.

A. R. Rees, M. Gregoriou, P. Johnson, and P. B. Garland, EMBO J. 3, 1843 (1984).
[PubMed]

Jones, G. R.

M. Martin-Fernandez, D. T. Clarke, M. J. Tobin, S. V. Jones, and G. R. Jones, Biophys. J. 82, 2415 (2002).
[CrossRef] [PubMed]

Jones, S. V.

M. Martin-Fernandez, D. T. Clarke, M. J. Tobin, S. V. Jones, and G. R. Jones, Biophys. J. 82, 2415 (2002).
[CrossRef] [PubMed]

Juskaitis, R.

Kusba, J.

J. Malicka, I. Gryczynski, J. Y. Fang, J. Kusba, and J. R. Lakowicz, J. Fluoresc. 12, 439 (2002).
[CrossRef]

Lakowicz, J. R.

J. Malicka, I. Gryczynski, J. Y. Fang, J. Kusba, and J. R. Lakowicz, J. Fluoresc. 12, 439 (2002).
[CrossRef]

Lévêque-Fort, S.

Lever, M. J.

Lommerse, P. H. M.

L. Cognet, G. S. Harms, G. A. Blab, P. H. M. Lommerse, and T. Schmidt, Appl. Phys. Lett. 77, 4052 (2000).
[CrossRef]

Malicka, J.

J. Malicka, I. Gryczynski, J. Y. Fang, J. Kusba, and J. R. Lakowicz, J. Fluoresc. 12, 439 (2002).
[CrossRef]

Martin-Fernandez, M.

M. Martin-Fernandez, D. T. Clarke, M. J. Tobin, S. V. Jones, and G. R. Jones, Biophys. J. 82, 2415 (2002).
[CrossRef] [PubMed]

Mattheyses, A. L.

A. L. Mattheyses, A. D. Hoppe, and D. Axelrod, Biophys. J. 87, 2787 (2004).
[CrossRef] [PubMed]

Minoghchi, S.

Y. Sako, S. Minoghchi, and T. Yanagida, Nat. Cell Biol. 2, 168 (2000).
[CrossRef] [PubMed]

Neil, M. A. A.

Parsons-Karavassilis, D.

Quinlan, M. E.

J. N. Forkey, M. E. Quinlan, and Y. E. Goldman, Biophys. J. 89, 1261 (2005).
[CrossRef] [PubMed]

Rees, A. R.

A. R. Rees, M. Gregoriou, P. Johnson, and P. B. Garland, EMBO J. 3, 1843 (1984).
[PubMed]

Sako, Y.

Y. Sako, S. Minoghchi, and T. Yanagida, Nat. Cell Biol. 2, 168 (2000).
[CrossRef] [PubMed]

Schmidt, T.

L. Cognet, G. S. Harms, G. A. Blab, P. H. M. Lommerse, and T. Schmidt, Appl. Phys. Lett. 77, 4052 (2000).
[CrossRef]

Siegel, J.

Stryer, L.

L. Stryer and R. P. Haugland, Proc. Natl. Acad. Sci. U.S.A. 58, 720 (1967).
[CrossRef]

Sucharov, L. O.

Tobin, M. J.

M. Martin-Fernandez, D. T. Clarke, M. J. Tobin, S. V. Jones, and G. R. Jones, Biophys. J. 82, 2415 (2002).
[CrossRef] [PubMed]

Wakelin, S.

S. Wakelin and C. R. Bagshaw, J. Microsc. 209, 143 (2003).
[CrossRef] [PubMed]

Webb, S. E. D.

Wilson, T.

Yanagida, T.

Y. Sako, S. Minoghchi, and T. Yanagida, Nat. Cell Biol. 2, 168 (2000).
[CrossRef] [PubMed]

Appl. Phys. Lett.

L. Cognet, G. S. Harms, G. A. Blab, P. H. M. Lommerse, and T. Schmidt, Appl. Phys. Lett. 77, 4052 (2000).
[CrossRef]

Biophys. J.

J. N. Forkey, M. E. Quinlan, and Y. E. Goldman, Biophys. J. 89, 1261 (2005).
[CrossRef] [PubMed]

A. L. Mattheyses, A. D. Hoppe, and D. Axelrod, Biophys. J. 87, 2787 (2004).
[CrossRef] [PubMed]

M. Martin-Fernandez, D. T. Clarke, M. J. Tobin, S. V. Jones, and G. R. Jones, Biophys. J. 82, 2415 (2002).
[CrossRef] [PubMed]

EMBO J.

A. R. Rees, M. Gregoriou, P. Johnson, and P. B. Garland, EMBO J. 3, 1843 (1984).
[PubMed]

J. Fluoresc.

J. Malicka, I. Gryczynski, J. Y. Fang, J. Kusba, and J. R. Lakowicz, J. Fluoresc. 12, 439 (2002).
[CrossRef]

J. Microsc.

S. Wakelin and C. R. Bagshaw, J. Microsc. 209, 143 (2003).
[CrossRef] [PubMed]

Methods

T. Ha, Methods 25, 78 (2001).
[CrossRef] [PubMed]

Methods Cell Biol.

D. Axelrod, Methods Cell Biol. 30, 245 (1989).
[CrossRef] [PubMed]

Nat. Cell Biol.

Y. Sako, S. Minoghchi, and T. Yanagida, Nat. Cell Biol. 2, 168 (2000).
[CrossRef] [PubMed]

Opt. Lett.

Proc. Natl. Acad. Sci. U.S.A.

L. Stryer and R. P. Haugland, Proc. Natl. Acad. Sci. U.S.A. 58, 720 (1967).
[CrossRef]

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Figures (3)

Fig. 1
Fig. 1

Experimental setup for multidimensional single-molecule fluorescence imaging. The Quad-View optics splits the fluorescence into four spatially identical images on the CCD, which differ solely in their spectral and polarization properties. A typical image of single fluorescent molecules in the membrane of live cells is shown, in this case murine EGF-Cy3 and murine EGF-Cy5, which appear on the left and right halves of the image, respectively. (Double-headed arrows indicate the axis of fluorescence polarization.)

Fig. 2
Fig. 2

Temporal variation of polarization-resolved intensity from a single Alexa 555-human EGF-Alexa 647 complex adherent on glass.

Fig. 3
Fig. 3

Polarization-resolved single-pair FRET in live cells. (a) Temporal variation of polarization and wavelength-resolved intensity from the Cy3 and Cy5 molecules indicated by a gray arrow in Fig. 1; (b) corresponding variation in FRET efficiency.

Equations (1)

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E = ( 1 + I D Φ A I A Φ D ) 1

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