Abstract

We describe instrumentation for real-time detection of single-molecule fluorescence in guided streams of 1-µm (nominal) water droplets. In this technique, target molecules were confined to droplets whose volumes were comparable with illumination volumes in diffraction-limited fluorescence microscopy and guided to the waist of a cw probe laser with an electrostatic potential. Concentration detection limits for Rhodamine 6G in water were determined to be ∼1 fM, roughly 3 orders of magnitude lower than corresponding limits determined recently with diffraction-limited microscopy techniques for a chemical separation of similar dyes. In addition to its utility as a vehicle for probing single molecules, instrumentation for producing and focusing stable streams of 1–2-µm-diameter droplets may have other important analytical applications as well.

© 1999 Optical Society of America

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  1. T. Hirschfeld, “Optical microscopic observation of single small molecules,” Appl. Opt. 15, 2965–2966 (1976).
    [CrossRef] [PubMed]
  2. E. B. Shera, N. K. Seitzinger, L. M. Davis, R. A. Keller, S. A. Soper, “Detection of single fluorescent molecules,” Chem. Phys. Lett. 174, 553–557 (1990).
    [CrossRef]
  3. For recent reviews see S. M. Nie, R. N. Zare, “Optical detection of single molecules,” Ann. Rev. Biophys. Biomol. Struct. 26, 567–596 (1997); R. A. Keller, W. P. Ambrose, P. M. Goodwin, J. H. Jett, J. C. Martin, M. Wu, “Single molecule fluorescence analysis in solution,” Appl. Spectrosc. 50, A12–A32 (1996); P. M. Goodwin, W. P. Ambrose, R. A. Keller, “Single-molecule detection in liquids by laser-induced fluorescence,” Acc. Chem. Res. 29, 607–613 (1996); and references cited therein.
  4. L. Q. Li, L. M. Davis, “Single-photon avalanche diode for single-molecule detection,” Rev. Sci. Instrum. 64, 1524–1529 (1993); “Rapid and efficient detection of single chromophore molecules in aqueous solution,” Appl. Opt. 34, 3208–3217 (1995).
    [CrossRef] [PubMed]
  5. Y. H. Lee, R. G. Maus, B. G. Smith, J. D. Winefordner, “Laser-induced fluorescence detection of a single molecule in a capillary,” Anal. Chem. 66, 4142–4149 (1994).
    [CrossRef]
  6. M. Sauer, K. H. Drexhage, C. Zander, J. Wolfrum, “Diode laser based detection of single molecules in solution,” Chem. Phys. Lett. 254, 223–228 (1996); C. Zander, K. H. Drexhage, K. T. Han, J. Wolfrum, M. Sauer, “Single-molecule counting and identification in a microcapillary,” Chem. Phys. Lett. 286, 457–465 (1998).
    [CrossRef]
  7. R. M. Dickson, D. J. Norris, Y.-L. Tzeng, W. E. Moerner, “Three-dimensional imaging of single molecules solvated in pores of poly(acrylamide) gels,” Science 274, 966–969 (1996).
    [CrossRef] [PubMed]
  8. X. Xu, E. S. Yeung, “Direct measurement of single-molecule diffusion and photodecomposition in free solution,” Science 275, 1106–1109 (1997).
    [CrossRef] [PubMed]
  9. C. Eggeling, J. Widengren, R. Rigler, C. A. M. Seidel, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: evidence of two-step photolysis,” Anal. Chem. 70, 2651–2659 (1998); J. Widengren, U. Mets, R. J. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution—a theoretical and experimental study,” J. Phys. Chem. 99, 13368–13379 (1995); M. Eigen, R. Rigler, “Sorting single molecules: applications to diagnostics and evolutionary biotechnology,” Proc. Natl. Acad. Sci. USA 91, 5740–5747 (1994).
    [CrossRef] [PubMed]
  10. S. Nie, D. T. Chiu, R. N. Zare, “Probing individual molecules with confocal fluorescence microscopy,” Science 266, 1018–1021 (1994); S. Nie, R. N. Zare, “Real-time detection of single-molecules in solution by confocal fluorescence microscopy,” Anal. Chem. 67, 2849–2857 (1995).
    [CrossRef] [PubMed]
  11. J. C. Fister, S. C. Jacobson, L. M. Davis, J. M. Ramsey, “Counting single chromophore molecules for ultrasensitive analysis and separations on microchip devices,” Anal. Chem. 70, 431–437 (1998).
    [CrossRef] [PubMed]
  12. M. D. Barnes, C-Y. Kung, W. B. Whitten, J. M. Ramsey, S. Arnold, S. Holler, “Fluorescence of oriented molecules in a microcavity,” Phys. Rev. Lett. 76, 3931–3934 (1996).
    [CrossRef] [PubMed]
  13. See R. K. Chang, A. J. Campillo, eds. Optical Effects in Microcavities (World Scientific, Singapore, 1996).
  14. M. D. Barnes, K. C. Ng, W. B. Whitten, J. M. Ramsey, “Detection of single rhodamine 6G molecules in levitated microdroplets,” Anal. Chem. 65, 2360–2365 (1993).
    [CrossRef]
  15. N. Lermer, M. D. Barnes, C-Y. Kung, W. B. Whitten, J. M. Ramsey, “High-efficiency molecular counting in solution: single-molecule detection in electrodynamically focused microdroplet streams,” Anal. Chem. 69, 2115–2121 (1997); M. D. Barnes, N. Lermer, C-Y. Kung, W. B. Whitten, J. M. Ramsey, “Real-time observation of single molecule fluorescence in microdroplet streams,” Opt. Lett. 22, 1265–1267 (1997); S. C. Hill, M. D. Barnes, N. Lermer, W. B. Whitten, J. M. Ramsey, “Simulation of single-molecule photocount statistics in microdroplets,” Anal. Chem. 70, 2964–2971 (1998).
    [CrossRef] [PubMed]
  16. S. A. Soper, H. L. Nutter, R. A. Keller, L. M. Davis, E. B. Shera, “The photophysical constants of several fluroescent dyes pertaining to ultrasensitive fluorescence,” Photochem. Photobiol. 57, 972–977 (1993).
    [CrossRef]
  17. C-Y. Kung, M. D. Barnes, N. Lermer, W. B. Whitten, J. M. Ramsey, “Confinement and manipulation of individual molecules in attoliter volumes,” Anal. Chem. 70, 658–661 (1998).
    [CrossRef] [PubMed]
  18. M. D. Barnes, N. Lermer, W. B. Whitten, J. M. Ramsey, “A CCD-based approach to high-precision size and refractive index determination of levitated microdroplets using Fraunhofer diffraction,” Rev. Sci. Instrum. 68, 2287–2291 (1997).
    [CrossRef]
  19. See, for example, N. A. Fuchs, Evaporation and Droplet Growth in Gaseous Media (Pergamon, New York, 1965).

1998

C. Eggeling, J. Widengren, R. Rigler, C. A. M. Seidel, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: evidence of two-step photolysis,” Anal. Chem. 70, 2651–2659 (1998); J. Widengren, U. Mets, R. J. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution—a theoretical and experimental study,” J. Phys. Chem. 99, 13368–13379 (1995); M. Eigen, R. Rigler, “Sorting single molecules: applications to diagnostics and evolutionary biotechnology,” Proc. Natl. Acad. Sci. USA 91, 5740–5747 (1994).
[CrossRef] [PubMed]

J. C. Fister, S. C. Jacobson, L. M. Davis, J. M. Ramsey, “Counting single chromophore molecules for ultrasensitive analysis and separations on microchip devices,” Anal. Chem. 70, 431–437 (1998).
[CrossRef] [PubMed]

C-Y. Kung, M. D. Barnes, N. Lermer, W. B. Whitten, J. M. Ramsey, “Confinement and manipulation of individual molecules in attoliter volumes,” Anal. Chem. 70, 658–661 (1998).
[CrossRef] [PubMed]

1997

M. D. Barnes, N. Lermer, W. B. Whitten, J. M. Ramsey, “A CCD-based approach to high-precision size and refractive index determination of levitated microdroplets using Fraunhofer diffraction,” Rev. Sci. Instrum. 68, 2287–2291 (1997).
[CrossRef]

N. Lermer, M. D. Barnes, C-Y. Kung, W. B. Whitten, J. M. Ramsey, “High-efficiency molecular counting in solution: single-molecule detection in electrodynamically focused microdroplet streams,” Anal. Chem. 69, 2115–2121 (1997); M. D. Barnes, N. Lermer, C-Y. Kung, W. B. Whitten, J. M. Ramsey, “Real-time observation of single molecule fluorescence in microdroplet streams,” Opt. Lett. 22, 1265–1267 (1997); S. C. Hill, M. D. Barnes, N. Lermer, W. B. Whitten, J. M. Ramsey, “Simulation of single-molecule photocount statistics in microdroplets,” Anal. Chem. 70, 2964–2971 (1998).
[CrossRef] [PubMed]

For recent reviews see S. M. Nie, R. N. Zare, “Optical detection of single molecules,” Ann. Rev. Biophys. Biomol. Struct. 26, 567–596 (1997); R. A. Keller, W. P. Ambrose, P. M. Goodwin, J. H. Jett, J. C. Martin, M. Wu, “Single molecule fluorescence analysis in solution,” Appl. Spectrosc. 50, A12–A32 (1996); P. M. Goodwin, W. P. Ambrose, R. A. Keller, “Single-molecule detection in liquids by laser-induced fluorescence,” Acc. Chem. Res. 29, 607–613 (1996); and references cited therein.

X. Xu, E. S. Yeung, “Direct measurement of single-molecule diffusion and photodecomposition in free solution,” Science 275, 1106–1109 (1997).
[CrossRef] [PubMed]

1996

M. Sauer, K. H. Drexhage, C. Zander, J. Wolfrum, “Diode laser based detection of single molecules in solution,” Chem. Phys. Lett. 254, 223–228 (1996); C. Zander, K. H. Drexhage, K. T. Han, J. Wolfrum, M. Sauer, “Single-molecule counting and identification in a microcapillary,” Chem. Phys. Lett. 286, 457–465 (1998).
[CrossRef]

R. M. Dickson, D. J. Norris, Y.-L. Tzeng, W. E. Moerner, “Three-dimensional imaging of single molecules solvated in pores of poly(acrylamide) gels,” Science 274, 966–969 (1996).
[CrossRef] [PubMed]

M. D. Barnes, C-Y. Kung, W. B. Whitten, J. M. Ramsey, S. Arnold, S. Holler, “Fluorescence of oriented molecules in a microcavity,” Phys. Rev. Lett. 76, 3931–3934 (1996).
[CrossRef] [PubMed]

1994

S. Nie, D. T. Chiu, R. N. Zare, “Probing individual molecules with confocal fluorescence microscopy,” Science 266, 1018–1021 (1994); S. Nie, R. N. Zare, “Real-time detection of single-molecules in solution by confocal fluorescence microscopy,” Anal. Chem. 67, 2849–2857 (1995).
[CrossRef] [PubMed]

Y. H. Lee, R. G. Maus, B. G. Smith, J. D. Winefordner, “Laser-induced fluorescence detection of a single molecule in a capillary,” Anal. Chem. 66, 4142–4149 (1994).
[CrossRef]

1993

L. Q. Li, L. M. Davis, “Single-photon avalanche diode for single-molecule detection,” Rev. Sci. Instrum. 64, 1524–1529 (1993); “Rapid and efficient detection of single chromophore molecules in aqueous solution,” Appl. Opt. 34, 3208–3217 (1995).
[CrossRef] [PubMed]

M. D. Barnes, K. C. Ng, W. B. Whitten, J. M. Ramsey, “Detection of single rhodamine 6G molecules in levitated microdroplets,” Anal. Chem. 65, 2360–2365 (1993).
[CrossRef]

S. A. Soper, H. L. Nutter, R. A. Keller, L. M. Davis, E. B. Shera, “The photophysical constants of several fluroescent dyes pertaining to ultrasensitive fluorescence,” Photochem. Photobiol. 57, 972–977 (1993).
[CrossRef]

1990

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

1976

Arnold, S.

M. D. Barnes, C-Y. Kung, W. B. Whitten, J. M. Ramsey, S. Arnold, S. Holler, “Fluorescence of oriented molecules in a microcavity,” Phys. Rev. Lett. 76, 3931–3934 (1996).
[CrossRef] [PubMed]

Barnes, M. D.

C-Y. Kung, M. D. Barnes, N. Lermer, W. B. Whitten, J. M. Ramsey, “Confinement and manipulation of individual molecules in attoliter volumes,” Anal. Chem. 70, 658–661 (1998).
[CrossRef] [PubMed]

M. D. Barnes, N. Lermer, W. B. Whitten, J. M. Ramsey, “A CCD-based approach to high-precision size and refractive index determination of levitated microdroplets using Fraunhofer diffraction,” Rev. Sci. Instrum. 68, 2287–2291 (1997).
[CrossRef]

N. Lermer, M. D. Barnes, C-Y. Kung, W. B. Whitten, J. M. Ramsey, “High-efficiency molecular counting in solution: single-molecule detection in electrodynamically focused microdroplet streams,” Anal. Chem. 69, 2115–2121 (1997); M. D. Barnes, N. Lermer, C-Y. Kung, W. B. Whitten, J. M. Ramsey, “Real-time observation of single molecule fluorescence in microdroplet streams,” Opt. Lett. 22, 1265–1267 (1997); S. C. Hill, M. D. Barnes, N. Lermer, W. B. Whitten, J. M. Ramsey, “Simulation of single-molecule photocount statistics in microdroplets,” Anal. Chem. 70, 2964–2971 (1998).
[CrossRef] [PubMed]

M. D. Barnes, C-Y. Kung, W. B. Whitten, J. M. Ramsey, S. Arnold, S. Holler, “Fluorescence of oriented molecules in a microcavity,” Phys. Rev. Lett. 76, 3931–3934 (1996).
[CrossRef] [PubMed]

M. D. Barnes, K. C. Ng, W. B. Whitten, J. M. Ramsey, “Detection of single rhodamine 6G molecules in levitated microdroplets,” Anal. Chem. 65, 2360–2365 (1993).
[CrossRef]

Chiu, D. T.

S. Nie, D. T. Chiu, R. N. Zare, “Probing individual molecules with confocal fluorescence microscopy,” Science 266, 1018–1021 (1994); S. Nie, R. N. Zare, “Real-time detection of single-molecules in solution by confocal fluorescence microscopy,” Anal. Chem. 67, 2849–2857 (1995).
[CrossRef] [PubMed]

Davis, L. M.

J. C. Fister, S. C. Jacobson, L. M. Davis, J. M. Ramsey, “Counting single chromophore molecules for ultrasensitive analysis and separations on microchip devices,” Anal. Chem. 70, 431–437 (1998).
[CrossRef] [PubMed]

L. Q. Li, L. M. Davis, “Single-photon avalanche diode for single-molecule detection,” Rev. Sci. Instrum. 64, 1524–1529 (1993); “Rapid and efficient detection of single chromophore molecules in aqueous solution,” Appl. Opt. 34, 3208–3217 (1995).
[CrossRef] [PubMed]

S. A. Soper, H. L. Nutter, R. A. Keller, L. M. Davis, E. B. Shera, “The photophysical constants of several fluroescent dyes pertaining to ultrasensitive fluorescence,” Photochem. Photobiol. 57, 972–977 (1993).
[CrossRef]

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

Dickson, R. M.

R. M. Dickson, D. J. Norris, Y.-L. Tzeng, W. E. Moerner, “Three-dimensional imaging of single molecules solvated in pores of poly(acrylamide) gels,” Science 274, 966–969 (1996).
[CrossRef] [PubMed]

Drexhage, K. H.

M. Sauer, K. H. Drexhage, C. Zander, J. Wolfrum, “Diode laser based detection of single molecules in solution,” Chem. Phys. Lett. 254, 223–228 (1996); C. Zander, K. H. Drexhage, K. T. Han, J. Wolfrum, M. Sauer, “Single-molecule counting and identification in a microcapillary,” Chem. Phys. Lett. 286, 457–465 (1998).
[CrossRef]

Eggeling, C.

C. Eggeling, J. Widengren, R. Rigler, C. A. M. Seidel, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: evidence of two-step photolysis,” Anal. Chem. 70, 2651–2659 (1998); J. Widengren, U. Mets, R. J. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution—a theoretical and experimental study,” J. Phys. Chem. 99, 13368–13379 (1995); M. Eigen, R. Rigler, “Sorting single molecules: applications to diagnostics and evolutionary biotechnology,” Proc. Natl. Acad. Sci. USA 91, 5740–5747 (1994).
[CrossRef] [PubMed]

Fister, J. C.

J. C. Fister, S. C. Jacobson, L. M. Davis, J. M. Ramsey, “Counting single chromophore molecules for ultrasensitive analysis and separations on microchip devices,” Anal. Chem. 70, 431–437 (1998).
[CrossRef] [PubMed]

Fuchs, N. A.

See, for example, N. A. Fuchs, Evaporation and Droplet Growth in Gaseous Media (Pergamon, New York, 1965).

Hirschfeld, T.

Holler, S.

M. D. Barnes, C-Y. Kung, W. B. Whitten, J. M. Ramsey, S. Arnold, S. Holler, “Fluorescence of oriented molecules in a microcavity,” Phys. Rev. Lett. 76, 3931–3934 (1996).
[CrossRef] [PubMed]

Jacobson, S. C.

J. C. Fister, S. C. Jacobson, L. M. Davis, J. M. Ramsey, “Counting single chromophore molecules for ultrasensitive analysis and separations on microchip devices,” Anal. Chem. 70, 431–437 (1998).
[CrossRef] [PubMed]

Keller, R. A.

S. A. Soper, H. L. Nutter, R. A. Keller, L. M. Davis, E. B. Shera, “The photophysical constants of several fluroescent dyes pertaining to ultrasensitive fluorescence,” Photochem. Photobiol. 57, 972–977 (1993).
[CrossRef]

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

Kung, C-Y.

C-Y. Kung, M. D. Barnes, N. Lermer, W. B. Whitten, J. M. Ramsey, “Confinement and manipulation of individual molecules in attoliter volumes,” Anal. Chem. 70, 658–661 (1998).
[CrossRef] [PubMed]

N. Lermer, M. D. Barnes, C-Y. Kung, W. B. Whitten, J. M. Ramsey, “High-efficiency molecular counting in solution: single-molecule detection in electrodynamically focused microdroplet streams,” Anal. Chem. 69, 2115–2121 (1997); M. D. Barnes, N. Lermer, C-Y. Kung, W. B. Whitten, J. M. Ramsey, “Real-time observation of single molecule fluorescence in microdroplet streams,” Opt. Lett. 22, 1265–1267 (1997); S. C. Hill, M. D. Barnes, N. Lermer, W. B. Whitten, J. M. Ramsey, “Simulation of single-molecule photocount statistics in microdroplets,” Anal. Chem. 70, 2964–2971 (1998).
[CrossRef] [PubMed]

M. D. Barnes, C-Y. Kung, W. B. Whitten, J. M. Ramsey, S. Arnold, S. Holler, “Fluorescence of oriented molecules in a microcavity,” Phys. Rev. Lett. 76, 3931–3934 (1996).
[CrossRef] [PubMed]

Lee, Y. H.

Y. H. Lee, R. G. Maus, B. G. Smith, J. D. Winefordner, “Laser-induced fluorescence detection of a single molecule in a capillary,” Anal. Chem. 66, 4142–4149 (1994).
[CrossRef]

Lermer, N.

C-Y. Kung, M. D. Barnes, N. Lermer, W. B. Whitten, J. M. Ramsey, “Confinement and manipulation of individual molecules in attoliter volumes,” Anal. Chem. 70, 658–661 (1998).
[CrossRef] [PubMed]

M. D. Barnes, N. Lermer, W. B. Whitten, J. M. Ramsey, “A CCD-based approach to high-precision size and refractive index determination of levitated microdroplets using Fraunhofer diffraction,” Rev. Sci. Instrum. 68, 2287–2291 (1997).
[CrossRef]

N. Lermer, M. D. Barnes, C-Y. Kung, W. B. Whitten, J. M. Ramsey, “High-efficiency molecular counting in solution: single-molecule detection in electrodynamically focused microdroplet streams,” Anal. Chem. 69, 2115–2121 (1997); M. D. Barnes, N. Lermer, C-Y. Kung, W. B. Whitten, J. M. Ramsey, “Real-time observation of single molecule fluorescence in microdroplet streams,” Opt. Lett. 22, 1265–1267 (1997); S. C. Hill, M. D. Barnes, N. Lermer, W. B. Whitten, J. M. Ramsey, “Simulation of single-molecule photocount statistics in microdroplets,” Anal. Chem. 70, 2964–2971 (1998).
[CrossRef] [PubMed]

Li, L. Q.

L. Q. Li, L. M. Davis, “Single-photon avalanche diode for single-molecule detection,” Rev. Sci. Instrum. 64, 1524–1529 (1993); “Rapid and efficient detection of single chromophore molecules in aqueous solution,” Appl. Opt. 34, 3208–3217 (1995).
[CrossRef] [PubMed]

Maus, R. G.

Y. H. Lee, R. G. Maus, B. G. Smith, J. D. Winefordner, “Laser-induced fluorescence detection of a single molecule in a capillary,” Anal. Chem. 66, 4142–4149 (1994).
[CrossRef]

Moerner, W. E.

R. M. Dickson, D. J. Norris, Y.-L. Tzeng, W. E. Moerner, “Three-dimensional imaging of single molecules solvated in pores of poly(acrylamide) gels,” Science 274, 966–969 (1996).
[CrossRef] [PubMed]

Ng, K. C.

M. D. Barnes, K. C. Ng, W. B. Whitten, J. M. Ramsey, “Detection of single rhodamine 6G molecules in levitated microdroplets,” Anal. Chem. 65, 2360–2365 (1993).
[CrossRef]

Nie, S.

S. Nie, D. T. Chiu, R. N. Zare, “Probing individual molecules with confocal fluorescence microscopy,” Science 266, 1018–1021 (1994); S. Nie, R. N. Zare, “Real-time detection of single-molecules in solution by confocal fluorescence microscopy,” Anal. Chem. 67, 2849–2857 (1995).
[CrossRef] [PubMed]

Nie, S. M.

For recent reviews see S. M. Nie, R. N. Zare, “Optical detection of single molecules,” Ann. Rev. Biophys. Biomol. Struct. 26, 567–596 (1997); R. A. Keller, W. P. Ambrose, P. M. Goodwin, J. H. Jett, J. C. Martin, M. Wu, “Single molecule fluorescence analysis in solution,” Appl. Spectrosc. 50, A12–A32 (1996); P. M. Goodwin, W. P. Ambrose, R. A. Keller, “Single-molecule detection in liquids by laser-induced fluorescence,” Acc. Chem. Res. 29, 607–613 (1996); and references cited therein.

Norris, D. J.

R. M. Dickson, D. J. Norris, Y.-L. Tzeng, W. E. Moerner, “Three-dimensional imaging of single molecules solvated in pores of poly(acrylamide) gels,” Science 274, 966–969 (1996).
[CrossRef] [PubMed]

Nutter, H. L.

S. A. Soper, H. L. Nutter, R. A. Keller, L. M. Davis, E. B. Shera, “The photophysical constants of several fluroescent dyes pertaining to ultrasensitive fluorescence,” Photochem. Photobiol. 57, 972–977 (1993).
[CrossRef]

Ramsey, J. M.

C-Y. Kung, M. D. Barnes, N. Lermer, W. B. Whitten, J. M. Ramsey, “Confinement and manipulation of individual molecules in attoliter volumes,” Anal. Chem. 70, 658–661 (1998).
[CrossRef] [PubMed]

J. C. Fister, S. C. Jacobson, L. M. Davis, J. M. Ramsey, “Counting single chromophore molecules for ultrasensitive analysis and separations on microchip devices,” Anal. Chem. 70, 431–437 (1998).
[CrossRef] [PubMed]

M. D. Barnes, N. Lermer, W. B. Whitten, J. M. Ramsey, “A CCD-based approach to high-precision size and refractive index determination of levitated microdroplets using Fraunhofer diffraction,” Rev. Sci. Instrum. 68, 2287–2291 (1997).
[CrossRef]

N. Lermer, M. D. Barnes, C-Y. Kung, W. B. Whitten, J. M. Ramsey, “High-efficiency molecular counting in solution: single-molecule detection in electrodynamically focused microdroplet streams,” Anal. Chem. 69, 2115–2121 (1997); M. D. Barnes, N. Lermer, C-Y. Kung, W. B. Whitten, J. M. Ramsey, “Real-time observation of single molecule fluorescence in microdroplet streams,” Opt. Lett. 22, 1265–1267 (1997); S. C. Hill, M. D. Barnes, N. Lermer, W. B. Whitten, J. M. Ramsey, “Simulation of single-molecule photocount statistics in microdroplets,” Anal. Chem. 70, 2964–2971 (1998).
[CrossRef] [PubMed]

M. D. Barnes, C-Y. Kung, W. B. Whitten, J. M. Ramsey, S. Arnold, S. Holler, “Fluorescence of oriented molecules in a microcavity,” Phys. Rev. Lett. 76, 3931–3934 (1996).
[CrossRef] [PubMed]

M. D. Barnes, K. C. Ng, W. B. Whitten, J. M. Ramsey, “Detection of single rhodamine 6G molecules in levitated microdroplets,” Anal. Chem. 65, 2360–2365 (1993).
[CrossRef]

Rigler, R.

C. Eggeling, J. Widengren, R. Rigler, C. A. M. Seidel, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: evidence of two-step photolysis,” Anal. Chem. 70, 2651–2659 (1998); J. Widengren, U. Mets, R. J. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution—a theoretical and experimental study,” J. Phys. Chem. 99, 13368–13379 (1995); M. Eigen, R. Rigler, “Sorting single molecules: applications to diagnostics and evolutionary biotechnology,” Proc. Natl. Acad. Sci. USA 91, 5740–5747 (1994).
[CrossRef] [PubMed]

Sauer, M.

M. Sauer, K. H. Drexhage, C. Zander, J. Wolfrum, “Diode laser based detection of single molecules in solution,” Chem. Phys. Lett. 254, 223–228 (1996); C. Zander, K. H. Drexhage, K. T. Han, J. Wolfrum, M. Sauer, “Single-molecule counting and identification in a microcapillary,” Chem. Phys. Lett. 286, 457–465 (1998).
[CrossRef]

Seidel, C. A. M.

C. Eggeling, J. Widengren, R. Rigler, C. A. M. Seidel, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: evidence of two-step photolysis,” Anal. Chem. 70, 2651–2659 (1998); J. Widengren, U. Mets, R. J. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution—a theoretical and experimental study,” J. Phys. Chem. 99, 13368–13379 (1995); M. Eigen, R. Rigler, “Sorting single molecules: applications to diagnostics and evolutionary biotechnology,” Proc. Natl. Acad. Sci. USA 91, 5740–5747 (1994).
[CrossRef] [PubMed]

Seitzinger, N. K.

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

Shera, E. B.

S. A. Soper, H. L. Nutter, R. A. Keller, L. M. Davis, E. B. Shera, “The photophysical constants of several fluroescent dyes pertaining to ultrasensitive fluorescence,” Photochem. Photobiol. 57, 972–977 (1993).
[CrossRef]

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

Smith, B. G.

Y. H. Lee, R. G. Maus, B. G. Smith, J. D. Winefordner, “Laser-induced fluorescence detection of a single molecule in a capillary,” Anal. Chem. 66, 4142–4149 (1994).
[CrossRef]

Soper, S. A.

S. A. Soper, H. L. Nutter, R. A. Keller, L. M. Davis, E. B. Shera, “The photophysical constants of several fluroescent dyes pertaining to ultrasensitive fluorescence,” Photochem. Photobiol. 57, 972–977 (1993).
[CrossRef]

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

Tzeng, Y.-L.

R. M. Dickson, D. J. Norris, Y.-L. Tzeng, W. E. Moerner, “Three-dimensional imaging of single molecules solvated in pores of poly(acrylamide) gels,” Science 274, 966–969 (1996).
[CrossRef] [PubMed]

Whitten, W. B.

C-Y. Kung, M. D. Barnes, N. Lermer, W. B. Whitten, J. M. Ramsey, “Confinement and manipulation of individual molecules in attoliter volumes,” Anal. Chem. 70, 658–661 (1998).
[CrossRef] [PubMed]

M. D. Barnes, N. Lermer, W. B. Whitten, J. M. Ramsey, “A CCD-based approach to high-precision size and refractive index determination of levitated microdroplets using Fraunhofer diffraction,” Rev. Sci. Instrum. 68, 2287–2291 (1997).
[CrossRef]

N. Lermer, M. D. Barnes, C-Y. Kung, W. B. Whitten, J. M. Ramsey, “High-efficiency molecular counting in solution: single-molecule detection in electrodynamically focused microdroplet streams,” Anal. Chem. 69, 2115–2121 (1997); M. D. Barnes, N. Lermer, C-Y. Kung, W. B. Whitten, J. M. Ramsey, “Real-time observation of single molecule fluorescence in microdroplet streams,” Opt. Lett. 22, 1265–1267 (1997); S. C. Hill, M. D. Barnes, N. Lermer, W. B. Whitten, J. M. Ramsey, “Simulation of single-molecule photocount statistics in microdroplets,” Anal. Chem. 70, 2964–2971 (1998).
[CrossRef] [PubMed]

M. D. Barnes, C-Y. Kung, W. B. Whitten, J. M. Ramsey, S. Arnold, S. Holler, “Fluorescence of oriented molecules in a microcavity,” Phys. Rev. Lett. 76, 3931–3934 (1996).
[CrossRef] [PubMed]

M. D. Barnes, K. C. Ng, W. B. Whitten, J. M. Ramsey, “Detection of single rhodamine 6G molecules in levitated microdroplets,” Anal. Chem. 65, 2360–2365 (1993).
[CrossRef]

Widengren, J.

C. Eggeling, J. Widengren, R. Rigler, C. A. M. Seidel, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: evidence of two-step photolysis,” Anal. Chem. 70, 2651–2659 (1998); J. Widengren, U. Mets, R. J. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution—a theoretical and experimental study,” J. Phys. Chem. 99, 13368–13379 (1995); M. Eigen, R. Rigler, “Sorting single molecules: applications to diagnostics and evolutionary biotechnology,” Proc. Natl. Acad. Sci. USA 91, 5740–5747 (1994).
[CrossRef] [PubMed]

Winefordner, J. D.

Y. H. Lee, R. G. Maus, B. G. Smith, J. D. Winefordner, “Laser-induced fluorescence detection of a single molecule in a capillary,” Anal. Chem. 66, 4142–4149 (1994).
[CrossRef]

Wolfrum, J.

M. Sauer, K. H. Drexhage, C. Zander, J. Wolfrum, “Diode laser based detection of single molecules in solution,” Chem. Phys. Lett. 254, 223–228 (1996); C. Zander, K. H. Drexhage, K. T. Han, J. Wolfrum, M. Sauer, “Single-molecule counting and identification in a microcapillary,” Chem. Phys. Lett. 286, 457–465 (1998).
[CrossRef]

Xu, X.

X. Xu, E. S. Yeung, “Direct measurement of single-molecule diffusion and photodecomposition in free solution,” Science 275, 1106–1109 (1997).
[CrossRef] [PubMed]

Yeung, E. S.

X. Xu, E. S. Yeung, “Direct measurement of single-molecule diffusion and photodecomposition in free solution,” Science 275, 1106–1109 (1997).
[CrossRef] [PubMed]

Zander, C.

M. Sauer, K. H. Drexhage, C. Zander, J. Wolfrum, “Diode laser based detection of single molecules in solution,” Chem. Phys. Lett. 254, 223–228 (1996); C. Zander, K. H. Drexhage, K. T. Han, J. Wolfrum, M. Sauer, “Single-molecule counting and identification in a microcapillary,” Chem. Phys. Lett. 286, 457–465 (1998).
[CrossRef]

Zare, R. N.

For recent reviews see S. M. Nie, R. N. Zare, “Optical detection of single molecules,” Ann. Rev. Biophys. Biomol. Struct. 26, 567–596 (1997); R. A. Keller, W. P. Ambrose, P. M. Goodwin, J. H. Jett, J. C. Martin, M. Wu, “Single molecule fluorescence analysis in solution,” Appl. Spectrosc. 50, A12–A32 (1996); P. M. Goodwin, W. P. Ambrose, R. A. Keller, “Single-molecule detection in liquids by laser-induced fluorescence,” Acc. Chem. Res. 29, 607–613 (1996); and references cited therein.

S. Nie, D. T. Chiu, R. N. Zare, “Probing individual molecules with confocal fluorescence microscopy,” Science 266, 1018–1021 (1994); S. Nie, R. N. Zare, “Real-time detection of single-molecules in solution by confocal fluorescence microscopy,” Anal. Chem. 67, 2849–2857 (1995).
[CrossRef] [PubMed]

Anal. Chem.

Y. H. Lee, R. G. Maus, B. G. Smith, J. D. Winefordner, “Laser-induced fluorescence detection of a single molecule in a capillary,” Anal. Chem. 66, 4142–4149 (1994).
[CrossRef]

C. Eggeling, J. Widengren, R. Rigler, C. A. M. Seidel, “Photobleaching of fluorescent dyes under conditions used for single-molecule detection: evidence of two-step photolysis,” Anal. Chem. 70, 2651–2659 (1998); J. Widengren, U. Mets, R. J. Rigler, “Fluorescence correlation spectroscopy of triplet states in solution—a theoretical and experimental study,” J. Phys. Chem. 99, 13368–13379 (1995); M. Eigen, R. Rigler, “Sorting single molecules: applications to diagnostics and evolutionary biotechnology,” Proc. Natl. Acad. Sci. USA 91, 5740–5747 (1994).
[CrossRef] [PubMed]

J. C. Fister, S. C. Jacobson, L. M. Davis, J. M. Ramsey, “Counting single chromophore molecules for ultrasensitive analysis and separations on microchip devices,” Anal. Chem. 70, 431–437 (1998).
[CrossRef] [PubMed]

M. D. Barnes, K. C. Ng, W. B. Whitten, J. M. Ramsey, “Detection of single rhodamine 6G molecules in levitated microdroplets,” Anal. Chem. 65, 2360–2365 (1993).
[CrossRef]

N. Lermer, M. D. Barnes, C-Y. Kung, W. B. Whitten, J. M. Ramsey, “High-efficiency molecular counting in solution: single-molecule detection in electrodynamically focused microdroplet streams,” Anal. Chem. 69, 2115–2121 (1997); M. D. Barnes, N. Lermer, C-Y. Kung, W. B. Whitten, J. M. Ramsey, “Real-time observation of single molecule fluorescence in microdroplet streams,” Opt. Lett. 22, 1265–1267 (1997); S. C. Hill, M. D. Barnes, N. Lermer, W. B. Whitten, J. M. Ramsey, “Simulation of single-molecule photocount statistics in microdroplets,” Anal. Chem. 70, 2964–2971 (1998).
[CrossRef] [PubMed]

C-Y. Kung, M. D. Barnes, N. Lermer, W. B. Whitten, J. M. Ramsey, “Confinement and manipulation of individual molecules in attoliter volumes,” Anal. Chem. 70, 658–661 (1998).
[CrossRef] [PubMed]

Ann. Rev. Biophys. Biomol. Struct.

For recent reviews see S. M. Nie, R. N. Zare, “Optical detection of single molecules,” Ann. Rev. Biophys. Biomol. Struct. 26, 567–596 (1997); R. A. Keller, W. P. Ambrose, P. M. Goodwin, J. H. Jett, J. C. Martin, M. Wu, “Single molecule fluorescence analysis in solution,” Appl. Spectrosc. 50, A12–A32 (1996); P. M. Goodwin, W. P. Ambrose, R. A. Keller, “Single-molecule detection in liquids by laser-induced fluorescence,” Acc. Chem. Res. 29, 607–613 (1996); and references cited therein.

Appl. Opt.

Chem. Phys. Lett.

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

M. Sauer, K. H. Drexhage, C. Zander, J. Wolfrum, “Diode laser based detection of single molecules in solution,” Chem. Phys. Lett. 254, 223–228 (1996); C. Zander, K. H. Drexhage, K. T. Han, J. Wolfrum, M. Sauer, “Single-molecule counting and identification in a microcapillary,” Chem. Phys. Lett. 286, 457–465 (1998).
[CrossRef]

Photochem. Photobiol.

S. A. Soper, H. L. Nutter, R. A. Keller, L. M. Davis, E. B. Shera, “The photophysical constants of several fluroescent dyes pertaining to ultrasensitive fluorescence,” Photochem. Photobiol. 57, 972–977 (1993).
[CrossRef]

Phys. Rev. Lett.

M. D. Barnes, C-Y. Kung, W. B. Whitten, J. M. Ramsey, S. Arnold, S. Holler, “Fluorescence of oriented molecules in a microcavity,” Phys. Rev. Lett. 76, 3931–3934 (1996).
[CrossRef] [PubMed]

Rev. Sci. Instrum.

M. D. Barnes, N. Lermer, W. B. Whitten, J. M. Ramsey, “A CCD-based approach to high-precision size and refractive index determination of levitated microdroplets using Fraunhofer diffraction,” Rev. Sci. Instrum. 68, 2287–2291 (1997).
[CrossRef]

L. Q. Li, L. M. Davis, “Single-photon avalanche diode for single-molecule detection,” Rev. Sci. Instrum. 64, 1524–1529 (1993); “Rapid and efficient detection of single chromophore molecules in aqueous solution,” Appl. Opt. 34, 3208–3217 (1995).
[CrossRef] [PubMed]

Science

R. M. Dickson, D. J. Norris, Y.-L. Tzeng, W. E. Moerner, “Three-dimensional imaging of single molecules solvated in pores of poly(acrylamide) gels,” Science 274, 966–969 (1996).
[CrossRef] [PubMed]

X. Xu, E. S. Yeung, “Direct measurement of single-molecule diffusion and photodecomposition in free solution,” Science 275, 1106–1109 (1997).
[CrossRef] [PubMed]

S. Nie, D. T. Chiu, R. N. Zare, “Probing individual molecules with confocal fluorescence microscopy,” Science 266, 1018–1021 (1994); S. Nie, R. N. Zare, “Real-time detection of single-molecules in solution by confocal fluorescence microscopy,” Anal. Chem. 67, 2849–2857 (1995).
[CrossRef] [PubMed]

Other

See R. K. Chang, A. J. Campillo, eds. Optical Effects in Microcavities (World Scientific, Singapore, 1996).

See, for example, N. A. Fuchs, Evaporation and Droplet Growth in Gaseous Media (Pergamon, New York, 1965).

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Figures (6)

Fig. 1
Fig. 1

Schematic of small-droplet generator. The left inset shows time and amplitude characteristics of typical applied-voltage pulses, and the right inset shows a bright-field micrograph of the Pyrex tip near the orifice. BOE, buffered oxide etchant.

Fig. 2
Fig. 2

Schematic of experimental apparatus used to probe single molecules in 1-µm-diameter water droplets. The inset shows a (nonscale) view of the droplet stream–laser intersection with typical experimental dimensions and parameters indicated. APD, avalanche photodiode; PMT, photomultiplier tube.

Fig. 3
Fig. 3

Segment of droplet fluorescence data from 16-pM R6G in (initial) 3.6-µm-diameter water droplets. The average number of molecules per droplet was 0.1 and the droplet size as probed was ∼1 µm.

Fig. 4
Fig. 4

Comparison of experimental blank (filled circles) and 16-pM R6G (open symbols) fluorescence amplitude distributions from droplets (12,000 measurements for each) with an initial diameter of 3.6 µm. The average numbers of blank background and single-molecule signal counts per droplet were 1.2 and 6.5, respectively; using a detection threshold of three counts gives a molecular detection probability of exp(-3/6.5) or 63%.

Fig. 5
Fig. 5

Comparison of 3000-shot segments of and 26-fM R6G droplets (≈9-µm initial diameter) probed with (a) 660-mW laser power, (b) 230-mW laser power, (c) blank (pure water) at 660 mW. The dashed lines indicate 99.8% confidence level (3s) detection thresholds. From the blank and fluorescence data, the concentration detection limit for this configuration was estimated to be 1 fM.

Fig. 6
Fig. 6

Histograms of measured counts for 660-mW (wide bars) and 230-mW (narrow bars) laser powers. A total of 30,000 droplets was used to construct the distribution for each intensity. The dashed curve shows a superposition of Poisson background distribution and single exponential with an average number of counts of 6.5.

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