Abstract

We have extended our recent experiments in the detection of single fluorescent molecules in solution to the exploration of spectroscopy at the single-molecule level. As a first step we have developed a technique that can efficiently distinguish between two species of dye molecules on the basis of differences in their emission spectra. We have also demonstrated that another spectroscopic property, fluorescence lifetime, can be accurately determined at the single-molecule level. Spectroscopic properties can be used to identify fluorescent molecules and to reveal static or slowly varying aspects of the microenvironment of each molecule, thereby yielding information unavailable from bulk studies.

© 1992 Optical Society of America

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  1. G. S. Hurst, M. H. Nayfeh, and J. P. Young, “A demonstration of one-atom detection,” Appl. Phys. Lett. 30, 229–231 (1977).
    [Crossref]
  2. W. E. Moerner and L. Kador, “Optical detection and spectroscopy of single molecules in a solid,” Phys. Rev. Lett. 62, 2535–2538 (1989).
    [Crossref] [PubMed]
  3. E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper, “Detection of single fluorescent molecules,” Chem. Phys. Lett. 174, 553–557 (1990).
    [Crossref]
  4. J. H. Jett, R. A. Keller, J. C. Martin, B. L. Marrone, R. K. Moyzis, R. L. Ratliff, N. K. Seitzinger, E. B. Shera, and C. C. Stewart, “High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules,” J. Biomol. Struct. Dynam. 7, 301–309 (1989).
    [Crossref]
  5. S. A. Soper, L. M. Davis, F. R. Fairfield, M. L. Hammond, C. A. Harger, J. H. Jett, R. A. Keller, B. L. Marrone, J. C. Martin, H. L. Nutter, E. B. Shera, and D. J. Simpson, “Rapid DNA sequencing based upon single molecule detection,” Proc. Int. Soc. Opt. Eng. 1435, 168–178 (1991).
  6. T. Hirschfeld, “Optical microscopic observation of single small molecules,” Appl. Opt. 15, 2965–2966 (1976).
    [Crossref] [PubMed]
  7. T. Hirschfeld, “Quantum efficiency independence of the time integrated emission from a fluorescent molecule,” Appl. Opt. 15, 3135–3139 (1976).
    [Crossref] [PubMed]
  8. N. J. Dovichi, J. C. Martin, J. H. Jett, and R. A. Keller, “Attogram detection limit for aqueous dye samples by laser-induced fluorescence,” Science 219, 845–847 (1982).
    [Crossref]
  9. N. J. Dovichi, J. C. Martin, J. H. Jett, M. Trkula, and R. A. Keller, “Laser-induced fluorescence of flowing samples as an approach to single-molecule detection in liquids,” Anal. Chem. 56, 348–354 (1984).
    [Crossref] [PubMed]
  10. F. Zarrin and N. J. Dovichi, “Sub-picoliter detection with the sheath flow cuvette,” Anal. Chem. 57, 2690–2692 (1985).
    [Crossref]
  11. J. V. Watson, “A method for improving light collection by 600% from square cross section flow cytometer chambers,” Br. J. Cancer 51, 433–435 (1985).
  12. J. V. Watson and M. J. Walport, “Molecular calibration in flow cytometry with sub-attogram detection limit,” J. Immunol. Methods 93, 171–175 (1986).
    [Crossref] [PubMed]
  13. D. C. Nguyen, R. A. Keller, and M. Trkula, “Ultrasensitive laser-induced fluorescence detection in hydrodynamically focused flows,” J. Opt. Soc. Am. B 4, 138–143 (1987).
    [Crossref]
  14. Y. F. Cheng and N. J. Dovichi, “Subattomole amino acid analysis by capillary zone electrophoresis and laser-induced fluorescence,” Science 242, 562–564 (1988).
    [Crossref] [PubMed]
  15. S. Wu and N. J. Dovichi, “High-sensitivity fluorescence detector for fluorescein isothiocyanate derivatives of amino acids separated by capillary zone electrophoresis,” J. Chromatogr. 480, 141–155 (1989).
    [Crossref] [PubMed]
  16. Y. F. Cheng, S. Wu, D. Y. Chen, and N. J. Dovichi, “Interaction of capillary zone electrophoresis with a sheath flow cuvette detector,” Anal. Chem. 62, 496–503 (1990).
    [Crossref]
  17. K. Peck, L. Stryer, A. N. Glazer, and R. A. Mathies, “Single-molecule fluorescence detection: autocorrelation criterion and experimental realization with Phycoerythrin,” Proc. Natl. Acad. Sci. USA 86, 4087–4091 (1989).
  18. S. A. Soper, E. B. Shera, J. C. Martin, J. H. Jett, J. H. Hahn, H. L. Nutter, and R. A. Keller, “Single molecule detection of Rhodamine 6G in ethanolic solutions using continuous wave laser excitation,” Anal. Chem. 63, 432–437 (1991).
    [Crossref]
  19. W. B. Whitten, J. M. Ramsey, S. A. Arnold, and B. V. Bronk, “Single-molecule detection limits in levitated microdroplets,” Anal. Chem. 63, 1027–1031 (1991).
    [Crossref]
  20. J. H. Hahn, S. A. Soper, H. L. Nutter, J. C. Martin, J. H. Jett, and R. A. Keller, “Laser-induced fluorescence determination of Rhodamine 6G at 6 × 10−15M,” Appl. Spectrosc. 45, 743 (1991).
    [Crossref]
  21. H. Giloh and J. Sedat, “Fluorescent microscopy: reduced photobleaching of Rhodamine and fluorescein protein conjugates by n-propyl gallate,” Science 217, 1252 (1982).
    [Crossref] [PubMed]
  22. C. W. Wilkerson, Los Alamos National Laboratory, Los Alamos, N.M. 87545 (personal communication, November1991).

1991 (4)

S. A. Soper, L. M. Davis, F. R. Fairfield, M. L. Hammond, C. A. Harger, J. H. Jett, R. A. Keller, B. L. Marrone, J. C. Martin, H. L. Nutter, E. B. Shera, and D. J. Simpson, “Rapid DNA sequencing based upon single molecule detection,” Proc. Int. Soc. Opt. Eng. 1435, 168–178 (1991).

S. A. Soper, E. B. Shera, J. C. Martin, J. H. Jett, J. H. Hahn, H. L. Nutter, and R. A. Keller, “Single molecule detection of Rhodamine 6G in ethanolic solutions using continuous wave laser excitation,” Anal. Chem. 63, 432–437 (1991).
[Crossref]

W. B. Whitten, J. M. Ramsey, S. A. Arnold, and B. V. Bronk, “Single-molecule detection limits in levitated microdroplets,” Anal. Chem. 63, 1027–1031 (1991).
[Crossref]

J. H. Hahn, S. A. Soper, H. L. Nutter, J. C. Martin, J. H. Jett, and R. A. Keller, “Laser-induced fluorescence determination of Rhodamine 6G at 6 × 10−15M,” Appl. Spectrosc. 45, 743 (1991).
[Crossref]

1990 (2)

E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper, “Detection of single fluorescent molecules,” Chem. Phys. Lett. 174, 553–557 (1990).
[Crossref]

Y. F. Cheng, S. Wu, D. Y. Chen, and N. J. Dovichi, “Interaction of capillary zone electrophoresis with a sheath flow cuvette detector,” Anal. Chem. 62, 496–503 (1990).
[Crossref]

1989 (4)

K. Peck, L. Stryer, A. N. Glazer, and R. A. Mathies, “Single-molecule fluorescence detection: autocorrelation criterion and experimental realization with Phycoerythrin,” Proc. Natl. Acad. Sci. USA 86, 4087–4091 (1989).

S. Wu and N. J. Dovichi, “High-sensitivity fluorescence detector for fluorescein isothiocyanate derivatives of amino acids separated by capillary zone electrophoresis,” J. Chromatogr. 480, 141–155 (1989).
[Crossref] [PubMed]

J. H. Jett, R. A. Keller, J. C. Martin, B. L. Marrone, R. K. Moyzis, R. L. Ratliff, N. K. Seitzinger, E. B. Shera, and C. C. Stewart, “High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules,” J. Biomol. Struct. Dynam. 7, 301–309 (1989).
[Crossref]

W. E. Moerner and L. Kador, “Optical detection and spectroscopy of single molecules in a solid,” Phys. Rev. Lett. 62, 2535–2538 (1989).
[Crossref] [PubMed]

1988 (1)

Y. F. Cheng and N. J. Dovichi, “Subattomole amino acid analysis by capillary zone electrophoresis and laser-induced fluorescence,” Science 242, 562–564 (1988).
[Crossref] [PubMed]

1987 (1)

1986 (1)

J. V. Watson and M. J. Walport, “Molecular calibration in flow cytometry with sub-attogram detection limit,” J. Immunol. Methods 93, 171–175 (1986).
[Crossref] [PubMed]

1985 (2)

F. Zarrin and N. J. Dovichi, “Sub-picoliter detection with the sheath flow cuvette,” Anal. Chem. 57, 2690–2692 (1985).
[Crossref]

J. V. Watson, “A method for improving light collection by 600% from square cross section flow cytometer chambers,” Br. J. Cancer 51, 433–435 (1985).

1984 (1)

N. J. Dovichi, J. C. Martin, J. H. Jett, M. Trkula, and R. A. Keller, “Laser-induced fluorescence of flowing samples as an approach to single-molecule detection in liquids,” Anal. Chem. 56, 348–354 (1984).
[Crossref] [PubMed]

1982 (2)

N. J. Dovichi, J. C. Martin, J. H. Jett, and R. A. Keller, “Attogram detection limit for aqueous dye samples by laser-induced fluorescence,” Science 219, 845–847 (1982).
[Crossref]

H. Giloh and J. Sedat, “Fluorescent microscopy: reduced photobleaching of Rhodamine and fluorescein protein conjugates by n-propyl gallate,” Science 217, 1252 (1982).
[Crossref] [PubMed]

1977 (1)

G. S. Hurst, M. H. Nayfeh, and J. P. Young, “A demonstration of one-atom detection,” Appl. Phys. Lett. 30, 229–231 (1977).
[Crossref]

1976 (2)

Arnold, S. A.

W. B. Whitten, J. M. Ramsey, S. A. Arnold, and B. V. Bronk, “Single-molecule detection limits in levitated microdroplets,” Anal. Chem. 63, 1027–1031 (1991).
[Crossref]

Bronk, B. V.

W. B. Whitten, J. M. Ramsey, S. A. Arnold, and B. V. Bronk, “Single-molecule detection limits in levitated microdroplets,” Anal. Chem. 63, 1027–1031 (1991).
[Crossref]

Chen, D. Y.

Y. F. Cheng, S. Wu, D. Y. Chen, and N. J. Dovichi, “Interaction of capillary zone electrophoresis with a sheath flow cuvette detector,” Anal. Chem. 62, 496–503 (1990).
[Crossref]

Cheng, Y. F.

Y. F. Cheng, S. Wu, D. Y. Chen, and N. J. Dovichi, “Interaction of capillary zone electrophoresis with a sheath flow cuvette detector,” Anal. Chem. 62, 496–503 (1990).
[Crossref]

Y. F. Cheng and N. J. Dovichi, “Subattomole amino acid analysis by capillary zone electrophoresis and laser-induced fluorescence,” Science 242, 562–564 (1988).
[Crossref] [PubMed]

Davis, L. M.

S. A. Soper, L. M. Davis, F. R. Fairfield, M. L. Hammond, C. A. Harger, J. H. Jett, R. A. Keller, B. L. Marrone, J. C. Martin, H. L. Nutter, E. B. Shera, and D. J. Simpson, “Rapid DNA sequencing based upon single molecule detection,” Proc. Int. Soc. Opt. Eng. 1435, 168–178 (1991).

E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper, “Detection of single fluorescent molecules,” Chem. Phys. Lett. 174, 553–557 (1990).
[Crossref]

Dovichi, N. J.

Y. F. Cheng, S. Wu, D. Y. Chen, and N. J. Dovichi, “Interaction of capillary zone electrophoresis with a sheath flow cuvette detector,” Anal. Chem. 62, 496–503 (1990).
[Crossref]

S. Wu and N. J. Dovichi, “High-sensitivity fluorescence detector for fluorescein isothiocyanate derivatives of amino acids separated by capillary zone electrophoresis,” J. Chromatogr. 480, 141–155 (1989).
[Crossref] [PubMed]

Y. F. Cheng and N. J. Dovichi, “Subattomole amino acid analysis by capillary zone electrophoresis and laser-induced fluorescence,” Science 242, 562–564 (1988).
[Crossref] [PubMed]

F. Zarrin and N. J. Dovichi, “Sub-picoliter detection with the sheath flow cuvette,” Anal. Chem. 57, 2690–2692 (1985).
[Crossref]

N. J. Dovichi, J. C. Martin, J. H. Jett, M. Trkula, and R. A. Keller, “Laser-induced fluorescence of flowing samples as an approach to single-molecule detection in liquids,” Anal. Chem. 56, 348–354 (1984).
[Crossref] [PubMed]

N. J. Dovichi, J. C. Martin, J. H. Jett, and R. A. Keller, “Attogram detection limit for aqueous dye samples by laser-induced fluorescence,” Science 219, 845–847 (1982).
[Crossref]

Fairfield, F. R.

S. A. Soper, L. M. Davis, F. R. Fairfield, M. L. Hammond, C. A. Harger, J. H. Jett, R. A. Keller, B. L. Marrone, J. C. Martin, H. L. Nutter, E. B. Shera, and D. J. Simpson, “Rapid DNA sequencing based upon single molecule detection,” Proc. Int. Soc. Opt. Eng. 1435, 168–178 (1991).

Giloh, H.

H. Giloh and J. Sedat, “Fluorescent microscopy: reduced photobleaching of Rhodamine and fluorescein protein conjugates by n-propyl gallate,” Science 217, 1252 (1982).
[Crossref] [PubMed]

Glazer, A. N.

K. Peck, L. Stryer, A. N. Glazer, and R. A. Mathies, “Single-molecule fluorescence detection: autocorrelation criterion and experimental realization with Phycoerythrin,” Proc. Natl. Acad. Sci. USA 86, 4087–4091 (1989).

Hahn, J. H.

S. A. Soper, E. B. Shera, J. C. Martin, J. H. Jett, J. H. Hahn, H. L. Nutter, and R. A. Keller, “Single molecule detection of Rhodamine 6G in ethanolic solutions using continuous wave laser excitation,” Anal. Chem. 63, 432–437 (1991).
[Crossref]

J. H. Hahn, S. A. Soper, H. L. Nutter, J. C. Martin, J. H. Jett, and R. A. Keller, “Laser-induced fluorescence determination of Rhodamine 6G at 6 × 10−15M,” Appl. Spectrosc. 45, 743 (1991).
[Crossref]

Hammond, M. L.

S. A. Soper, L. M. Davis, F. R. Fairfield, M. L. Hammond, C. A. Harger, J. H. Jett, R. A. Keller, B. L. Marrone, J. C. Martin, H. L. Nutter, E. B. Shera, and D. J. Simpson, “Rapid DNA sequencing based upon single molecule detection,” Proc. Int. Soc. Opt. Eng. 1435, 168–178 (1991).

Harger, C. A.

S. A. Soper, L. M. Davis, F. R. Fairfield, M. L. Hammond, C. A. Harger, J. H. Jett, R. A. Keller, B. L. Marrone, J. C. Martin, H. L. Nutter, E. B. Shera, and D. J. Simpson, “Rapid DNA sequencing based upon single molecule detection,” Proc. Int. Soc. Opt. Eng. 1435, 168–178 (1991).

Hirschfeld, T.

Hurst, G. S.

G. S. Hurst, M. H. Nayfeh, and J. P. Young, “A demonstration of one-atom detection,” Appl. Phys. Lett. 30, 229–231 (1977).
[Crossref]

Jett, J. H.

S. A. Soper, L. M. Davis, F. R. Fairfield, M. L. Hammond, C. A. Harger, J. H. Jett, R. A. Keller, B. L. Marrone, J. C. Martin, H. L. Nutter, E. B. Shera, and D. J. Simpson, “Rapid DNA sequencing based upon single molecule detection,” Proc. Int. Soc. Opt. Eng. 1435, 168–178 (1991).

S. A. Soper, E. B. Shera, J. C. Martin, J. H. Jett, J. H. Hahn, H. L. Nutter, and R. A. Keller, “Single molecule detection of Rhodamine 6G in ethanolic solutions using continuous wave laser excitation,” Anal. Chem. 63, 432–437 (1991).
[Crossref]

J. H. Hahn, S. A. Soper, H. L. Nutter, J. C. Martin, J. H. Jett, and R. A. Keller, “Laser-induced fluorescence determination of Rhodamine 6G at 6 × 10−15M,” Appl. Spectrosc. 45, 743 (1991).
[Crossref]

J. H. Jett, R. A. Keller, J. C. Martin, B. L. Marrone, R. K. Moyzis, R. L. Ratliff, N. K. Seitzinger, E. B. Shera, and C. C. Stewart, “High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules,” J. Biomol. Struct. Dynam. 7, 301–309 (1989).
[Crossref]

N. J. Dovichi, J. C. Martin, J. H. Jett, M. Trkula, and R. A. Keller, “Laser-induced fluorescence of flowing samples as an approach to single-molecule detection in liquids,” Anal. Chem. 56, 348–354 (1984).
[Crossref] [PubMed]

N. J. Dovichi, J. C. Martin, J. H. Jett, and R. A. Keller, “Attogram detection limit for aqueous dye samples by laser-induced fluorescence,” Science 219, 845–847 (1982).
[Crossref]

Kador, L.

W. E. Moerner and L. Kador, “Optical detection and spectroscopy of single molecules in a solid,” Phys. Rev. Lett. 62, 2535–2538 (1989).
[Crossref] [PubMed]

Keller, R. A.

S. A. Soper, L. M. Davis, F. R. Fairfield, M. L. Hammond, C. A. Harger, J. H. Jett, R. A. Keller, B. L. Marrone, J. C. Martin, H. L. Nutter, E. B. Shera, and D. J. Simpson, “Rapid DNA sequencing based upon single molecule detection,” Proc. Int. Soc. Opt. Eng. 1435, 168–178 (1991).

S. A. Soper, E. B. Shera, J. C. Martin, J. H. Jett, J. H. Hahn, H. L. Nutter, and R. A. Keller, “Single molecule detection of Rhodamine 6G in ethanolic solutions using continuous wave laser excitation,” Anal. Chem. 63, 432–437 (1991).
[Crossref]

J. H. Hahn, S. A. Soper, H. L. Nutter, J. C. Martin, J. H. Jett, and R. A. Keller, “Laser-induced fluorescence determination of Rhodamine 6G at 6 × 10−15M,” Appl. Spectrosc. 45, 743 (1991).
[Crossref]

E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper, “Detection of single fluorescent molecules,” Chem. Phys. Lett. 174, 553–557 (1990).
[Crossref]

J. H. Jett, R. A. Keller, J. C. Martin, B. L. Marrone, R. K. Moyzis, R. L. Ratliff, N. K. Seitzinger, E. B. Shera, and C. C. Stewart, “High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules,” J. Biomol. Struct. Dynam. 7, 301–309 (1989).
[Crossref]

D. C. Nguyen, R. A. Keller, and M. Trkula, “Ultrasensitive laser-induced fluorescence detection in hydrodynamically focused flows,” J. Opt. Soc. Am. B 4, 138–143 (1987).
[Crossref]

N. J. Dovichi, J. C. Martin, J. H. Jett, M. Trkula, and R. A. Keller, “Laser-induced fluorescence of flowing samples as an approach to single-molecule detection in liquids,” Anal. Chem. 56, 348–354 (1984).
[Crossref] [PubMed]

N. J. Dovichi, J. C. Martin, J. H. Jett, and R. A. Keller, “Attogram detection limit for aqueous dye samples by laser-induced fluorescence,” Science 219, 845–847 (1982).
[Crossref]

Marrone, B. L.

S. A. Soper, L. M. Davis, F. R. Fairfield, M. L. Hammond, C. A. Harger, J. H. Jett, R. A. Keller, B. L. Marrone, J. C. Martin, H. L. Nutter, E. B. Shera, and D. J. Simpson, “Rapid DNA sequencing based upon single molecule detection,” Proc. Int. Soc. Opt. Eng. 1435, 168–178 (1991).

J. H. Jett, R. A. Keller, J. C. Martin, B. L. Marrone, R. K. Moyzis, R. L. Ratliff, N. K. Seitzinger, E. B. Shera, and C. C. Stewart, “High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules,” J. Biomol. Struct. Dynam. 7, 301–309 (1989).
[Crossref]

Martin, J. C.

S. A. Soper, L. M. Davis, F. R. Fairfield, M. L. Hammond, C. A. Harger, J. H. Jett, R. A. Keller, B. L. Marrone, J. C. Martin, H. L. Nutter, E. B. Shera, and D. J. Simpson, “Rapid DNA sequencing based upon single molecule detection,” Proc. Int. Soc. Opt. Eng. 1435, 168–178 (1991).

S. A. Soper, E. B. Shera, J. C. Martin, J. H. Jett, J. H. Hahn, H. L. Nutter, and R. A. Keller, “Single molecule detection of Rhodamine 6G in ethanolic solutions using continuous wave laser excitation,” Anal. Chem. 63, 432–437 (1991).
[Crossref]

J. H. Hahn, S. A. Soper, H. L. Nutter, J. C. Martin, J. H. Jett, and R. A. Keller, “Laser-induced fluorescence determination of Rhodamine 6G at 6 × 10−15M,” Appl. Spectrosc. 45, 743 (1991).
[Crossref]

J. H. Jett, R. A. Keller, J. C. Martin, B. L. Marrone, R. K. Moyzis, R. L. Ratliff, N. K. Seitzinger, E. B. Shera, and C. C. Stewart, “High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules,” J. Biomol. Struct. Dynam. 7, 301–309 (1989).
[Crossref]

N. J. Dovichi, J. C. Martin, J. H. Jett, M. Trkula, and R. A. Keller, “Laser-induced fluorescence of flowing samples as an approach to single-molecule detection in liquids,” Anal. Chem. 56, 348–354 (1984).
[Crossref] [PubMed]

N. J. Dovichi, J. C. Martin, J. H. Jett, and R. A. Keller, “Attogram detection limit for aqueous dye samples by laser-induced fluorescence,” Science 219, 845–847 (1982).
[Crossref]

Mathies, R. A.

K. Peck, L. Stryer, A. N. Glazer, and R. A. Mathies, “Single-molecule fluorescence detection: autocorrelation criterion and experimental realization with Phycoerythrin,” Proc. Natl. Acad. Sci. USA 86, 4087–4091 (1989).

Moerner, W. E.

W. E. Moerner and L. Kador, “Optical detection and spectroscopy of single molecules in a solid,” Phys. Rev. Lett. 62, 2535–2538 (1989).
[Crossref] [PubMed]

Moyzis, R. K.

J. H. Jett, R. A. Keller, J. C. Martin, B. L. Marrone, R. K. Moyzis, R. L. Ratliff, N. K. Seitzinger, E. B. Shera, and C. C. Stewart, “High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules,” J. Biomol. Struct. Dynam. 7, 301–309 (1989).
[Crossref]

Nayfeh, M. H.

G. S. Hurst, M. H. Nayfeh, and J. P. Young, “A demonstration of one-atom detection,” Appl. Phys. Lett. 30, 229–231 (1977).
[Crossref]

Nguyen, D. C.

Nutter, H. L.

S. A. Soper, E. B. Shera, J. C. Martin, J. H. Jett, J. H. Hahn, H. L. Nutter, and R. A. Keller, “Single molecule detection of Rhodamine 6G in ethanolic solutions using continuous wave laser excitation,” Anal. Chem. 63, 432–437 (1991).
[Crossref]

S. A. Soper, L. M. Davis, F. R. Fairfield, M. L. Hammond, C. A. Harger, J. H. Jett, R. A. Keller, B. L. Marrone, J. C. Martin, H. L. Nutter, E. B. Shera, and D. J. Simpson, “Rapid DNA sequencing based upon single molecule detection,” Proc. Int. Soc. Opt. Eng. 1435, 168–178 (1991).

J. H. Hahn, S. A. Soper, H. L. Nutter, J. C. Martin, J. H. Jett, and R. A. Keller, “Laser-induced fluorescence determination of Rhodamine 6G at 6 × 10−15M,” Appl. Spectrosc. 45, 743 (1991).
[Crossref]

Peck, K.

K. Peck, L. Stryer, A. N. Glazer, and R. A. Mathies, “Single-molecule fluorescence detection: autocorrelation criterion and experimental realization with Phycoerythrin,” Proc. Natl. Acad. Sci. USA 86, 4087–4091 (1989).

Ramsey, J. M.

W. B. Whitten, J. M. Ramsey, S. A. Arnold, and B. V. Bronk, “Single-molecule detection limits in levitated microdroplets,” Anal. Chem. 63, 1027–1031 (1991).
[Crossref]

Ratliff, R. L.

J. H. Jett, R. A. Keller, J. C. Martin, B. L. Marrone, R. K. Moyzis, R. L. Ratliff, N. K. Seitzinger, E. B. Shera, and C. C. Stewart, “High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules,” J. Biomol. Struct. Dynam. 7, 301–309 (1989).
[Crossref]

Sedat, J.

H. Giloh and J. Sedat, “Fluorescent microscopy: reduced photobleaching of Rhodamine and fluorescein protein conjugates by n-propyl gallate,” Science 217, 1252 (1982).
[Crossref] [PubMed]

Seitzinger, N. K.

E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper, “Detection of single fluorescent molecules,” Chem. Phys. Lett. 174, 553–557 (1990).
[Crossref]

J. H. Jett, R. A. Keller, J. C. Martin, B. L. Marrone, R. K. Moyzis, R. L. Ratliff, N. K. Seitzinger, E. B. Shera, and C. C. Stewart, “High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules,” J. Biomol. Struct. Dynam. 7, 301–309 (1989).
[Crossref]

Shera, E. B.

S. A. Soper, L. M. Davis, F. R. Fairfield, M. L. Hammond, C. A. Harger, J. H. Jett, R. A. Keller, B. L. Marrone, J. C. Martin, H. L. Nutter, E. B. Shera, and D. J. Simpson, “Rapid DNA sequencing based upon single molecule detection,” Proc. Int. Soc. Opt. Eng. 1435, 168–178 (1991).

S. A. Soper, E. B. Shera, J. C. Martin, J. H. Jett, J. H. Hahn, H. L. Nutter, and R. A. Keller, “Single molecule detection of Rhodamine 6G in ethanolic solutions using continuous wave laser excitation,” Anal. Chem. 63, 432–437 (1991).
[Crossref]

E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper, “Detection of single fluorescent molecules,” Chem. Phys. Lett. 174, 553–557 (1990).
[Crossref]

J. H. Jett, R. A. Keller, J. C. Martin, B. L. Marrone, R. K. Moyzis, R. L. Ratliff, N. K. Seitzinger, E. B. Shera, and C. C. Stewart, “High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules,” J. Biomol. Struct. Dynam. 7, 301–309 (1989).
[Crossref]

Simpson, D. J.

S. A. Soper, L. M. Davis, F. R. Fairfield, M. L. Hammond, C. A. Harger, J. H. Jett, R. A. Keller, B. L. Marrone, J. C. Martin, H. L. Nutter, E. B. Shera, and D. J. Simpson, “Rapid DNA sequencing based upon single molecule detection,” Proc. Int. Soc. Opt. Eng. 1435, 168–178 (1991).

Soper, S. A.

S. A. Soper, E. B. Shera, J. C. Martin, J. H. Jett, J. H. Hahn, H. L. Nutter, and R. A. Keller, “Single molecule detection of Rhodamine 6G in ethanolic solutions using continuous wave laser excitation,” Anal. Chem. 63, 432–437 (1991).
[Crossref]

S. A. Soper, L. M. Davis, F. R. Fairfield, M. L. Hammond, C. A. Harger, J. H. Jett, R. A. Keller, B. L. Marrone, J. C. Martin, H. L. Nutter, E. B. Shera, and D. J. Simpson, “Rapid DNA sequencing based upon single molecule detection,” Proc. Int. Soc. Opt. Eng. 1435, 168–178 (1991).

J. H. Hahn, S. A. Soper, H. L. Nutter, J. C. Martin, J. H. Jett, and R. A. Keller, “Laser-induced fluorescence determination of Rhodamine 6G at 6 × 10−15M,” Appl. Spectrosc. 45, 743 (1991).
[Crossref]

E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper, “Detection of single fluorescent molecules,” Chem. Phys. Lett. 174, 553–557 (1990).
[Crossref]

Stewart, C. C.

J. H. Jett, R. A. Keller, J. C. Martin, B. L. Marrone, R. K. Moyzis, R. L. Ratliff, N. K. Seitzinger, E. B. Shera, and C. C. Stewart, “High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules,” J. Biomol. Struct. Dynam. 7, 301–309 (1989).
[Crossref]

Stryer, L.

K. Peck, L. Stryer, A. N. Glazer, and R. A. Mathies, “Single-molecule fluorescence detection: autocorrelation criterion and experimental realization with Phycoerythrin,” Proc. Natl. Acad. Sci. USA 86, 4087–4091 (1989).

Trkula, M.

D. C. Nguyen, R. A. Keller, and M. Trkula, “Ultrasensitive laser-induced fluorescence detection in hydrodynamically focused flows,” J. Opt. Soc. Am. B 4, 138–143 (1987).
[Crossref]

N. J. Dovichi, J. C. Martin, J. H. Jett, M. Trkula, and R. A. Keller, “Laser-induced fluorescence of flowing samples as an approach to single-molecule detection in liquids,” Anal. Chem. 56, 348–354 (1984).
[Crossref] [PubMed]

Walport, M. J.

J. V. Watson and M. J. Walport, “Molecular calibration in flow cytometry with sub-attogram detection limit,” J. Immunol. Methods 93, 171–175 (1986).
[Crossref] [PubMed]

Watson, J. V.

J. V. Watson and M. J. Walport, “Molecular calibration in flow cytometry with sub-attogram detection limit,” J. Immunol. Methods 93, 171–175 (1986).
[Crossref] [PubMed]

J. V. Watson, “A method for improving light collection by 600% from square cross section flow cytometer chambers,” Br. J. Cancer 51, 433–435 (1985).

Whitten, W. B.

W. B. Whitten, J. M. Ramsey, S. A. Arnold, and B. V. Bronk, “Single-molecule detection limits in levitated microdroplets,” Anal. Chem. 63, 1027–1031 (1991).
[Crossref]

Wilkerson, C. W.

C. W. Wilkerson, Los Alamos National Laboratory, Los Alamos, N.M. 87545 (personal communication, November1991).

Wu, S.

Y. F. Cheng, S. Wu, D. Y. Chen, and N. J. Dovichi, “Interaction of capillary zone electrophoresis with a sheath flow cuvette detector,” Anal. Chem. 62, 496–503 (1990).
[Crossref]

S. Wu and N. J. Dovichi, “High-sensitivity fluorescence detector for fluorescein isothiocyanate derivatives of amino acids separated by capillary zone electrophoresis,” J. Chromatogr. 480, 141–155 (1989).
[Crossref] [PubMed]

Young, J. P.

G. S. Hurst, M. H. Nayfeh, and J. P. Young, “A demonstration of one-atom detection,” Appl. Phys. Lett. 30, 229–231 (1977).
[Crossref]

Zarrin, F.

F. Zarrin and N. J. Dovichi, “Sub-picoliter detection with the sheath flow cuvette,” Anal. Chem. 57, 2690–2692 (1985).
[Crossref]

Anal. Chem. (5)

N. J. Dovichi, J. C. Martin, J. H. Jett, M. Trkula, and R. A. Keller, “Laser-induced fluorescence of flowing samples as an approach to single-molecule detection in liquids,” Anal. Chem. 56, 348–354 (1984).
[Crossref] [PubMed]

F. Zarrin and N. J. Dovichi, “Sub-picoliter detection with the sheath flow cuvette,” Anal. Chem. 57, 2690–2692 (1985).
[Crossref]

Y. F. Cheng, S. Wu, D. Y. Chen, and N. J. Dovichi, “Interaction of capillary zone electrophoresis with a sheath flow cuvette detector,” Anal. Chem. 62, 496–503 (1990).
[Crossref]

S. A. Soper, E. B. Shera, J. C. Martin, J. H. Jett, J. H. Hahn, H. L. Nutter, and R. A. Keller, “Single molecule detection of Rhodamine 6G in ethanolic solutions using continuous wave laser excitation,” Anal. Chem. 63, 432–437 (1991).
[Crossref]

W. B. Whitten, J. M. Ramsey, S. A. Arnold, and B. V. Bronk, “Single-molecule detection limits in levitated microdroplets,” Anal. Chem. 63, 1027–1031 (1991).
[Crossref]

Appl. Opt. (2)

Appl. Phys. Lett. (1)

G. S. Hurst, M. H. Nayfeh, and J. P. Young, “A demonstration of one-atom detection,” Appl. Phys. Lett. 30, 229–231 (1977).
[Crossref]

Appl. Spectrosc. (1)

Br. J. Cancer (1)

J. V. Watson, “A method for improving light collection by 600% from square cross section flow cytometer chambers,” Br. J. Cancer 51, 433–435 (1985).

Chem. Phys. Lett. (1)

E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, and S. A. Soper, “Detection of single fluorescent molecules,” Chem. Phys. Lett. 174, 553–557 (1990).
[Crossref]

J. Biomol. Struct. Dynam. (1)

J. H. Jett, R. A. Keller, J. C. Martin, B. L. Marrone, R. K. Moyzis, R. L. Ratliff, N. K. Seitzinger, E. B. Shera, and C. C. Stewart, “High-speed DNA sequencing: an approach based upon fluorescence detection of single molecules,” J. Biomol. Struct. Dynam. 7, 301–309 (1989).
[Crossref]

J. Chromatogr. (1)

S. Wu and N. J. Dovichi, “High-sensitivity fluorescence detector for fluorescein isothiocyanate derivatives of amino acids separated by capillary zone electrophoresis,” J. Chromatogr. 480, 141–155 (1989).
[Crossref] [PubMed]

J. Immunol. Methods (1)

J. V. Watson and M. J. Walport, “Molecular calibration in flow cytometry with sub-attogram detection limit,” J. Immunol. Methods 93, 171–175 (1986).
[Crossref] [PubMed]

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

Phys. Rev. Lett. (1)

W. E. Moerner and L. Kador, “Optical detection and spectroscopy of single molecules in a solid,” Phys. Rev. Lett. 62, 2535–2538 (1989).
[Crossref] [PubMed]

Proc. Int. Soc. Opt. Eng. (1)

S. A. Soper, L. M. Davis, F. R. Fairfield, M. L. Hammond, C. A. Harger, J. H. Jett, R. A. Keller, B. L. Marrone, J. C. Martin, H. L. Nutter, E. B. Shera, and D. J. Simpson, “Rapid DNA sequencing based upon single molecule detection,” Proc. Int. Soc. Opt. Eng. 1435, 168–178 (1991).

Proc. Natl. Acad. Sci. USA (1)

K. Peck, L. Stryer, A. N. Glazer, and R. A. Mathies, “Single-molecule fluorescence detection: autocorrelation criterion and experimental realization with Phycoerythrin,” Proc. Natl. Acad. Sci. USA 86, 4087–4091 (1989).

Science (3)

N. J. Dovichi, J. C. Martin, J. H. Jett, and R. A. Keller, “Attogram detection limit for aqueous dye samples by laser-induced fluorescence,” Science 219, 845–847 (1982).
[Crossref]

H. Giloh and J. Sedat, “Fluorescent microscopy: reduced photobleaching of Rhodamine and fluorescein protein conjugates by n-propyl gallate,” Science 217, 1252 (1982).
[Crossref] [PubMed]

Y. F. Cheng and N. J. Dovichi, “Subattomole amino acid analysis by capillary zone electrophoresis and laser-induced fluorescence,” Science 242, 562–564 (1988).
[Crossref] [PubMed]

Other (1)

C. W. Wilkerson, Los Alamos National Laboratory, Los Alamos, N.M. 87545 (personal communication, November1991).

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Figures (11)

Fig. 1
Fig. 1

Block diagram of the single-molecule detection and identification apparatus: λ/2, half-wave plate; BS1, 70%/30% beam splitter; LP1, lens pair for adjustment of size and divergence of the 532-nm beam; BS2, 4%/96% beam splitter for diverting a small percentage of the 532-nm beam to the triggering photodiode; A1, beam attenuator for 532 nm; DF1, dichroic mirror for directing and combining excitation beams; LP2, lens pair used for adjustment of the size and the divergence of the 585-nm beam; A2, beam attenuator for 585 nm; L1, laser focusing lens; DF2, dichroic mirror for separating the fluorescence of the dyes; BP1, BP2, bandpass filters; ADC, analog-to-digital converter; PMT’s, photomultipliers.

Fig. 2
Fig. 2

Fluorescence emission spectra of R6G and TR (thinner solid curves), the transmission spectra of the fluorescence isolation dichroic filter DF2 (heavy solid curve), and the fluorescence bandpass filters (dashed and dotted–dashed curves).

Fig. 3
Fig. 3

Time spectra for CH3OH from a, detector 1, and b, detector 2. Spectrum a was shifted upward for clarity. The time window in which the fluorescent photons were recorded by the multichannel scalers is shown. These windows contained approximately 60% of the photons available from the entire decay. The small bump near 1.5 ns is caused by reflected light.

Fig. 4
Fig. 4

Autocorrelation functions for CH3OH, 1 × 10−14 M R6G and 1 × 10−14 M TR solutions for detectors 1 and 2. The average laser power used in these experiments was 30 mW each for 532- and 585-nm excitation. The data were collected in 1-ms time widths (204,800 total data points) and then binned by 4 before analysis. The A(0) points are off scale.

Fig. 5
Fig. 5

Weighted-quadratic-sum plots for a mixed 1 × 10−14 M methanol solution of TR and R6G (top) and TR alone (bottom) from detector 1, the R6G channel. At 10−14 M approximately one dye molecule enters the detection volume every 4 s.

Fig. 6
Fig. 6

Weighted-quadratic-sum plots for a mixed 1 × 10−14 M methanol solution of TR and R6G (top) and R6G alone (bottom) from detector 2, the TR channel.

Fig. 7
Fig. 7

Autocorrelation plots for water and for mixed 5 × 10−14 M R6G and TR in water, either O2 saturated (no purge) or deoxygenated (purged), for both detectors. In both plots the purged data are represented by triangles.

Fig. 8
Fig. 8

Weighted-quadratic-sum plots for a mixed 5 × 10−14 M O2-purged water solution of R6G and TR (top) and purged water alone (bottom) from detector 1. For the water data the value of k = 9 was used in the weighted-quadratic-sum filter procedure. At 5 × 10−14 M approximately one dye molecule passes through the detector every second.

Fig. 9
Fig. 9

Weighted-quadratic-sum plots for a mixed 5 × 10−14 M, O2-purged water solution of R6G and TR (top) and purged water alone (bottom) from detector 2. See the caption of Fig. 8.

Fig. 10
Fig. 10

Arrival time profile for the burst of photons from a single molecule. The time scale on the horizontal axis has an arbitrary origin; this particular burst occurred ∼15.5 s after the start of the experiment. An irregular multiple-peaked profile such as this is often seen when a molecule diffuses into, out of, and back into the detection volume. A total of 215 photons was detected from this particular burst, of which fewer than 10 were background.

Fig. 11
Fig. 11

Estimating the lifetime of a single molecule. The decay time (difference between time of the laser pulse and time of photon arrival) associated with each photon detected during the burst shown in Fig. 10 was histogrammed to produce the noisy exponential decay profile shown. From this data set the maximum-likelihood estimate of the lifetime of this particular molecule was 4.5 ± 0.3 ns, in agreement with the value 4.17 ± 0.01 ns determined by measurements on bulk solutions (see text). Lifetimes computed for other individual bursts were also clustered near the bulk value. The two bins near t = 0 are far off scale because they include prompt Raman-scattered photons. (Only delayed events were included in the profile shown in Fig. 10.)

Equations (2)

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A ( τ ) = t = 0 N 1 d ( t ) d ( t + τ ) .
S ( t ) = τ = 0 k 1 w ( τ ) d ( t + τ ) 2 .

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