Abstract

We present a technique for observing single fluorophore molecules in solution. A mode-locked laser beam is focused into the solution, thereby defining a two-photon excitation volume localized in three dimensions. Molecules diffusing into and out of this volume produce fluorescence bursts, which are detected with a high signal-to-background ratio. The theoretical foundations for the technique are laid out, including the attendant fluorescence rate distribution, and agree with experimental results obtained for Rhodamine B molecules in water.

© 1995 Optical Society of America

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  1. D. C. Nguyen, R. A. Keller, J. H. Jett, J. C. Martin, Anal. Chem. 59, 2158 (1987).
    [CrossRef] [PubMed]
  2. K. Peck, L. Stryer, A. N. Glazer, R. A. Mathies, Proc. Natl. Acad. Sci. (USA) 86, 4087 (1989).
    [CrossRef] [PubMed]
  3. L.-Q. Li, L. M. Davis, Rev. Sci. Instrum. 64, 1524 (1993).
    [CrossRef]
  4. S. A. Soper, Q. L. Mattingly, P. Vegunta, Anal. Chem. 66, 740 (1993).
    [CrossRef]
  5. Y.-H. Lee, R. G. Maus, B. W. Smith, J. D. Winefordner, Anal. Chem. 66, 4142 (1994).
    [CrossRef]
  6. M. D. Barnes, K. C. Ng, W. B. Whitten, J. M. Ramsey, Anal. Chem. 65, 2360 (1993).
    [CrossRef]
  7. Ü. Mets, R. Rigler, J. Fluoresc. 4, 259 (1994).
    [CrossRef]
  8. S. Nie, D. T. Chiu, R. N. Zare, Science 266, 1018 (1994).
    [CrossRef] [PubMed]
  9. W. Denk, J. H. Strickler, W. W. Webb, Science 248, 73 (1990).
    [CrossRef] [PubMed]
  10. W. W. Webb, C. Xu, “Measurement of two-photon excitation cross-sections of molecular fluorophores with data from 690 nm to 1050 nm” J. Opt. Soc. Am. B (to be published).
  11. K. M. Berland, P. T. C. So, E. Gratton, Biophys. J. 68, 694 (1995).
    [CrossRef] [PubMed]
  12. D. Madge, E. L. Elson, W. W. Webb, Phys. Rev. Lett. 29, 705 (1972).
    [CrossRef]
  13. N. L. Thompson, in Topics in Fluorescence Spectroscopy, Vol. 1: Techniques, J. R. Lakowicz, ed. (Plenum, New York, 1991), p. 337.
  14. M. Eigen, R. Rigler, Proc. Natl. Acad. Sci. (USA) 91, 5740 (1994).
    [CrossRef] [PubMed]
  15. K. S. Wells, D. R. Sandison, J. Strickler, W. W. Webb, in Handbook of Biological Confocal Microscopy, J. Pawley, ed. (IMR, Madison, Wisc., 1989), p. 23.

1995

K. M. Berland, P. T. C. So, E. Gratton, Biophys. J. 68, 694 (1995).
[CrossRef] [PubMed]

1994

M. Eigen, R. Rigler, Proc. Natl. Acad. Sci. (USA) 91, 5740 (1994).
[CrossRef] [PubMed]

Y.-H. Lee, R. G. Maus, B. W. Smith, J. D. Winefordner, Anal. Chem. 66, 4142 (1994).
[CrossRef]

Ü. Mets, R. Rigler, J. Fluoresc. 4, 259 (1994).
[CrossRef]

S. Nie, D. T. Chiu, R. N. Zare, Science 266, 1018 (1994).
[CrossRef] [PubMed]

1993

M. D. Barnes, K. C. Ng, W. B. Whitten, J. M. Ramsey, Anal. Chem. 65, 2360 (1993).
[CrossRef]

L.-Q. Li, L. M. Davis, Rev. Sci. Instrum. 64, 1524 (1993).
[CrossRef]

S. A. Soper, Q. L. Mattingly, P. Vegunta, Anal. Chem. 66, 740 (1993).
[CrossRef]

1990

W. Denk, J. H. Strickler, W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

1989

K. Peck, L. Stryer, A. N. Glazer, R. A. Mathies, Proc. Natl. Acad. Sci. (USA) 86, 4087 (1989).
[CrossRef] [PubMed]

1987

D. C. Nguyen, R. A. Keller, J. H. Jett, J. C. Martin, Anal. Chem. 59, 2158 (1987).
[CrossRef] [PubMed]

1972

D. Madge, E. L. Elson, W. W. Webb, Phys. Rev. Lett. 29, 705 (1972).
[CrossRef]

Barnes, M. D.

M. D. Barnes, K. C. Ng, W. B. Whitten, J. M. Ramsey, Anal. Chem. 65, 2360 (1993).
[CrossRef]

Berland, K. M.

K. M. Berland, P. T. C. So, E. Gratton, Biophys. J. 68, 694 (1995).
[CrossRef] [PubMed]

Chiu, D. T.

S. Nie, D. T. Chiu, R. N. Zare, Science 266, 1018 (1994).
[CrossRef] [PubMed]

Davis, L. M.

L.-Q. Li, L. M. Davis, Rev. Sci. Instrum. 64, 1524 (1993).
[CrossRef]

Denk, W.

W. Denk, J. H. Strickler, W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Eigen, M.

M. Eigen, R. Rigler, Proc. Natl. Acad. Sci. (USA) 91, 5740 (1994).
[CrossRef] [PubMed]

Elson, E. L.

D. Madge, E. L. Elson, W. W. Webb, Phys. Rev. Lett. 29, 705 (1972).
[CrossRef]

Glazer, A. N.

K. Peck, L. Stryer, A. N. Glazer, R. A. Mathies, Proc. Natl. Acad. Sci. (USA) 86, 4087 (1989).
[CrossRef] [PubMed]

Gratton, E.

K. M. Berland, P. T. C. So, E. Gratton, Biophys. J. 68, 694 (1995).
[CrossRef] [PubMed]

Jett, J. H.

D. C. Nguyen, R. A. Keller, J. H. Jett, J. C. Martin, Anal. Chem. 59, 2158 (1987).
[CrossRef] [PubMed]

Keller, R. A.

D. C. Nguyen, R. A. Keller, J. H. Jett, J. C. Martin, Anal. Chem. 59, 2158 (1987).
[CrossRef] [PubMed]

Lee, Y.-H.

Y.-H. Lee, R. G. Maus, B. W. Smith, J. D. Winefordner, Anal. Chem. 66, 4142 (1994).
[CrossRef]

Li, L.-Q.

L.-Q. Li, L. M. Davis, Rev. Sci. Instrum. 64, 1524 (1993).
[CrossRef]

Madge, D.

D. Madge, E. L. Elson, W. W. Webb, Phys. Rev. Lett. 29, 705 (1972).
[CrossRef]

Martin, J. C.

D. C. Nguyen, R. A. Keller, J. H. Jett, J. C. Martin, Anal. Chem. 59, 2158 (1987).
[CrossRef] [PubMed]

Mathies, R. A.

K. Peck, L. Stryer, A. N. Glazer, R. A. Mathies, Proc. Natl. Acad. Sci. (USA) 86, 4087 (1989).
[CrossRef] [PubMed]

Mattingly, Q. L.

S. A. Soper, Q. L. Mattingly, P. Vegunta, Anal. Chem. 66, 740 (1993).
[CrossRef]

Maus, R. G.

Y.-H. Lee, R. G. Maus, B. W. Smith, J. D. Winefordner, Anal. Chem. 66, 4142 (1994).
[CrossRef]

Mets, Ü.

Ü. Mets, R. Rigler, J. Fluoresc. 4, 259 (1994).
[CrossRef]

Ng, K. C.

M. D. Barnes, K. C. Ng, W. B. Whitten, J. M. Ramsey, Anal. Chem. 65, 2360 (1993).
[CrossRef]

Nguyen, D. C.

D. C. Nguyen, R. A. Keller, J. H. Jett, J. C. Martin, Anal. Chem. 59, 2158 (1987).
[CrossRef] [PubMed]

Nie, S.

S. Nie, D. T. Chiu, R. N. Zare, Science 266, 1018 (1994).
[CrossRef] [PubMed]

Peck, K.

K. Peck, L. Stryer, A. N. Glazer, R. A. Mathies, Proc. Natl. Acad. Sci. (USA) 86, 4087 (1989).
[CrossRef] [PubMed]

Ramsey, J. M.

M. D. Barnes, K. C. Ng, W. B. Whitten, J. M. Ramsey, Anal. Chem. 65, 2360 (1993).
[CrossRef]

Rigler, R.

Ü. Mets, R. Rigler, J. Fluoresc. 4, 259 (1994).
[CrossRef]

M. Eigen, R. Rigler, Proc. Natl. Acad. Sci. (USA) 91, 5740 (1994).
[CrossRef] [PubMed]

Sandison, D. R.

K. S. Wells, D. R. Sandison, J. Strickler, W. W. Webb, in Handbook of Biological Confocal Microscopy, J. Pawley, ed. (IMR, Madison, Wisc., 1989), p. 23.

Smith, B. W.

Y.-H. Lee, R. G. Maus, B. W. Smith, J. D. Winefordner, Anal. Chem. 66, 4142 (1994).
[CrossRef]

So, P. T. C.

K. M. Berland, P. T. C. So, E. Gratton, Biophys. J. 68, 694 (1995).
[CrossRef] [PubMed]

Soper, S. A.

S. A. Soper, Q. L. Mattingly, P. Vegunta, Anal. Chem. 66, 740 (1993).
[CrossRef]

Strickler, J.

K. S. Wells, D. R. Sandison, J. Strickler, W. W. Webb, in Handbook of Biological Confocal Microscopy, J. Pawley, ed. (IMR, Madison, Wisc., 1989), p. 23.

Strickler, J. H.

W. Denk, J. H. Strickler, W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Stryer, L.

K. Peck, L. Stryer, A. N. Glazer, R. A. Mathies, Proc. Natl. Acad. Sci. (USA) 86, 4087 (1989).
[CrossRef] [PubMed]

Thompson, N. L.

N. L. Thompson, in Topics in Fluorescence Spectroscopy, Vol. 1: Techniques, J. R. Lakowicz, ed. (Plenum, New York, 1991), p. 337.

Vegunta, P.

S. A. Soper, Q. L. Mattingly, P. Vegunta, Anal. Chem. 66, 740 (1993).
[CrossRef]

Webb, W. W.

W. Denk, J. H. Strickler, W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

D. Madge, E. L. Elson, W. W. Webb, Phys. Rev. Lett. 29, 705 (1972).
[CrossRef]

K. S. Wells, D. R. Sandison, J. Strickler, W. W. Webb, in Handbook of Biological Confocal Microscopy, J. Pawley, ed. (IMR, Madison, Wisc., 1989), p. 23.

W. W. Webb, C. Xu, “Measurement of two-photon excitation cross-sections of molecular fluorophores with data from 690 nm to 1050 nm” J. Opt. Soc. Am. B (to be published).

Wells, K. S.

K. S. Wells, D. R. Sandison, J. Strickler, W. W. Webb, in Handbook of Biological Confocal Microscopy, J. Pawley, ed. (IMR, Madison, Wisc., 1989), p. 23.

Whitten, W. B.

M. D. Barnes, K. C. Ng, W. B. Whitten, J. M. Ramsey, Anal. Chem. 65, 2360 (1993).
[CrossRef]

Winefordner, J. D.

Y.-H. Lee, R. G. Maus, B. W. Smith, J. D. Winefordner, Anal. Chem. 66, 4142 (1994).
[CrossRef]

Xu, C.

W. W. Webb, C. Xu, “Measurement of two-photon excitation cross-sections of molecular fluorophores with data from 690 nm to 1050 nm” J. Opt. Soc. Am. B (to be published).

Zare, R. N.

S. Nie, D. T. Chiu, R. N. Zare, Science 266, 1018 (1994).
[CrossRef] [PubMed]

Anal. Chem.

S. A. Soper, Q. L. Mattingly, P. Vegunta, Anal. Chem. 66, 740 (1993).
[CrossRef]

Y.-H. Lee, R. G. Maus, B. W. Smith, J. D. Winefordner, Anal. Chem. 66, 4142 (1994).
[CrossRef]

M. D. Barnes, K. C. Ng, W. B. Whitten, J. M. Ramsey, Anal. Chem. 65, 2360 (1993).
[CrossRef]

D. C. Nguyen, R. A. Keller, J. H. Jett, J. C. Martin, Anal. Chem. 59, 2158 (1987).
[CrossRef] [PubMed]

Biophys. J.

K. M. Berland, P. T. C. So, E. Gratton, Biophys. J. 68, 694 (1995).
[CrossRef] [PubMed]

J. Fluoresc.

Ü. Mets, R. Rigler, J. Fluoresc. 4, 259 (1994).
[CrossRef]

Phys. Rev. Lett.

D. Madge, E. L. Elson, W. W. Webb, Phys. Rev. Lett. 29, 705 (1972).
[CrossRef]

Proc. Natl. Acad. Sci.

K. Peck, L. Stryer, A. N. Glazer, R. A. Mathies, Proc. Natl. Acad. Sci. (USA) 86, 4087 (1989).
[CrossRef] [PubMed]

M. Eigen, R. Rigler, Proc. Natl. Acad. Sci. (USA) 91, 5740 (1994).
[CrossRef] [PubMed]

Rev. Sci. Instrum.

L.-Q. Li, L. M. Davis, Rev. Sci. Instrum. 64, 1524 (1993).
[CrossRef]

Science

S. Nie, D. T. Chiu, R. N. Zare, Science 266, 1018 (1994).
[CrossRef] [PubMed]

W. Denk, J. H. Strickler, W. W. Webb, Science 248, 73 (1990).
[CrossRef] [PubMed]

Other

W. W. Webb, C. Xu, “Measurement of two-photon excitation cross-sections of molecular fluorophores with data from 690 nm to 1050 nm” J. Opt. Soc. Am. B (to be published).

N. L. Thompson, in Topics in Fluorescence Spectroscopy, Vol. 1: Techniques, J. R. Lakowicz, ed. (Plenum, New York, 1991), p. 337.

K. S. Wells, D. R. Sandison, J. Strickler, W. W. Webb, in Handbook of Biological Confocal Microscopy, J. Pawley, ed. (IMR, Madison, Wisc., 1989), p. 23.

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Figures (3)

Fig. 1
Fig. 1

A mode-locked laser beam is collimated (C), filtered (F), doubled in repetition rate (B’s are 50% beam splitters) and focused into a droplet of RB solution. The resultant fluorescence (dashed line) is epicollected through an interference filter (IF) and focused onto an avalanche photodiode (APD). NA is numerical aperture.

Fig. 2
Fig. 2

Measured autocorrelation trace for a 60-pM solution of RB (bin size 10 μs) and theoretical fit assuming a Gaussian–Lorentzian focus of waist 750 nm. The fit provides a RB diffusion constant (≃3 × 10−6 cm2/s, as expected) and a dwell time τd ≃ 0.7 ms. The measured autocorrelation trace for pure water is featureless.

Fig. 3
Fig. 3

Top: Fluorescence bursts from a 6-pM solution of RB (raw data, bin size 1.3 ms). The theoretical average burst height is 1.0, corresponding to a fluorescence rate of 8 × 105 s−1 at the sample. The burst rate is ~100 s−1. Bottom: Corresponding background from a blank sample (0.17 count/bin average).

Equations (7)

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

F ( t ) = α I 0 2 W 2 ( r ) C ( r , t ) d r ,
G ( τ ) = δ F ( t ) δ F ( t + τ ) ¯ / F ( t ) ¯ 2 ,
G ( 0 + ) = ξ 2 / N ¯ ,
V = γ - 1 W 2 ( r ) d r ,
τ d = G ( 0 + ) - 1 0 G ( τ ) d τ .
P ( f r 0 ) = w 0 2 sinh ( r r 0 / 2 D τ ) 8 f r 0 D τ exp [ - ( r 2 + r 0 2 ) / 4 D τ ] ,
D n 0 1 ( ξ f ) n e - ξ f n ! P ( f ) d f ,

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