Abstract

The physical and instrumental processes that occur in experiments for the detection of individual fluorescent molecules in solution are described, with emphasis on their incorporation into a quantitative Monte Carlo simulation. The simulation is applied to the conditions of a past experiment [Appl. Opt. 34, 3208 (1995)], which utilizes a sheath flow system for high detection efficiency, and it generates comparable results, while helping to identify experimental limitations. The simulation indicates that the use of low dead-time electronics and appropriate selection of experimental parameters should enable detection at more rapid rates for applications in which large numbers of molecules are to be efficiently counted.

© 1998 Optical Society of America

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  1. L. Q. Li, L. M. Davis, “Rapid and efficient detection of single chromophore molecules in aqueous solution,” Appl. Opt. 34, 3208–3217 (1995), and references cited therein.
    [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. K. Kneipp, Y. Wang, H. Kneipp, L. T. Perelman, I. Itzkan, R. R. Dasari, M. S. Feld, “Single molecule detection using surface-enhanced Raman scattering (SERS),” Phys. Rev. Lett. 78, 1667–1670 (1997).
    [Crossref]
  4. S. Nie, S. R. Emory, “Probing single nanoparticles by surface enhanced Raman scattering,” Science 275, 1102–1106 (1997).
    [Crossref] [PubMed]
  5. L. Q. Li, L. M. Davis, “Single photon avalanche diode for single molecule detection,” Rev. Sci. Instrum. 64, 1524–1529 (1993).
    [Crossref]
  6. S. A. Soper, Q. L. Mattingly, P. Vegunta, “Photon burst detection of single near-infrared fluorescent molecules,” Anal. Chem. 65, 740–747 (1993).
    [Crossref]
  7. Y. H. Lee, R. G. Maus, B. W. Smith, J. D. Winefordner, “Laser-induced fluorescence detection of a single molecule in a capillary,” Anal. Chem. 66, 4142–4149 (1994).
    [Crossref]
  8. M. Eigen, R. Rigler, “Sorting single molecules: application to diagnostics and evolutionary biotechnology,” Proc. Natl. Acad. Sci. USA 91, 5740–5747 (1994).
    [Crossref] [PubMed]
  9. S. Nie, D. T. Chiu, R. N. Zare, “Probing individual molecules with confocal fluorescence microscopy,” Science 266, 1018–1021 (1994).
    [Crossref] [PubMed]
  10. X. H. Xu, E. S. Yeung, “Direct measurement of single-molecule diffusion and photodecomposition in free solution,” Science 275, 1106–1109 (1997).
    [Crossref] [PubMed]
  11. J. Mertz, C. Xu, W. W. Webb, “Single-molecule detection by two-photon-excited fluorescence,” Opt. Lett. 20, 2532–2534 (1995).
    [Crossref] [PubMed]
  12. P. M. Goodwin, R. L. Affleck, W. P. Ambrose, J. H. Jett, M. E. Johnson, J. C. Martin, J. T. Petty, J. A. Schecker, M. Wu, R. A. Keller, “Detection of single fluorescent molecules in flowing sample streams,” in Computer Assisted Analytical Spectroscopy, S. D. Brown, ed. (Wiley, Sussex, England, 1996), pp. 61–80.
  13. S. Nie, D. T. Chiu, R. N. Zare, “Real-time detection of single molecules in solution by confocal fluorescence microscopy,” Anal. Chem. 67, 2849–2857 (1995).
    [Crossref]
  14. L. M. Davis, L. Q. Li, “Monte Carlo model of a single molecule counting experiment,” in Laser Applications to Chemical Analysis, Vol. 5 of 1994 OSA Technical Digest Series (Optical Society of America, Washington, D.C., 1994), pp. 206–209.
  15. R. A. Mathies, K. Peck, L. Stryer, “Optimization of high-sensitivity fluorescence detection,” Anal. Chem. 62, 1786–1791 (1990).
    [Crossref] [PubMed]
  16. L. M. Davis, L. Q. Li, E. B. Shera, A. Castro, S. A. Soper, “Photon statistics for the detection of single molecules in solution,” in Quantum Electronics and Laser Science, Vol. 13 of OSA Technical Digest Series (Optical Society of America (Washington, D.C., 1992), pp. 70–72.
  17. S. A. Soper, L. M. Davis, E. B. Shera, “Detection and identification of single molecules in solution,” J. Opt. Soc. Am. B 9, 1761–1769 (1992).
    [Crossref]
  18. C. Zander, M. Sauer, K. H. Drexage, D. S. Ko, A. Schultz, J. Wolfrum, L. Brand, C. Eggeling, C. A. M. Seidel, “Detection and characterization of single molecules in aqueous solution,” Appl. Phys. B 63, 517–523 (1996).
  19. D. H. Bunfield, “Simulation of a single molecule detection experiment,” M.S. thesis (University of Tennessee, Knoxville, Tennessee, 1997).
  20. L. M. Davis, D. H. Bunfield, “Spectroscopic identification of individually detected fluorescent molecules,” The Fifth International Conference on Methods and Applications of Fluorescence Spectroscopy, Berlin, 1997. Book of Abstracts, W. Rettig, ed. (Springer-Verlag, Berlin, 1997), p. 27.
  21. A. Spinelli, L. M. Davis, H. Dautet, “Actively quenched single-photon avalanche diode for high repetition rate time-gated photon counting,” Rev. Sci. Instrum. 67, 55–61 (1996).
    [Crossref]
  22. 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 (1997).
    [Crossref]
  23. One significant application currently under development that demands efficient and unambiguous SMD is rapid DNA sequencing. Also, efficient processing of the sample is desirable for assay of miniscule sample quantities, such as the components of a single cell and genetic screening without DNA amplification by polymerase chain reaction. See references of Ref. 1.
  24. R. Loudon, The Quantum Theory of Light, 2nd ed. (Oxford University, New York, 1973), pp. 78–81.
  25. A. Penzkofer, W. Falkenstein, W. Kaiser, “Vibronic relaxation in the S1 state of rhodamine dye solutions,” Chem. Phys. Lett. 44, 82–87 (1976).
    [Crossref]
  26. H. Qian, E. L. Elson, “Analysis of confocal laser-induced microscope optics for 3-D fluorescence correlation spectroscopy,” Appl. Opt. 30, 1185–1195 (1991).
    [Crossref] [PubMed]
  27. S. A. Soper, H. L. Nutter, R. A. Keller, L. M. Davis, E. B. Shera, “The photophysical constants of several fluorescent dyes pertaining to ultrasensitive fluorescence spectroscopy,” Photochem. Photobiol. 57, 972–977 (1993).
    [Crossref]
  28. L. M. Davis, L. E. Schneider, D. B. Bunfield, “Increasing the rate of detection of single molecules in solution,” in Laser Applications to Chemical, Biological, and Environmental Analysis, Vol. 3 of 1996 OSA Technical Digest Series (Optical Society of America, Washington, D.C., 1996), pp. 24–26.
  29. L. M. Davis, “Efficient counting of single molecules with sub-100 μs transit times,” paper BC.06 at American Physical Society SES97 meeting, Nashville, November 1997, http://aps.org/BAPSSES97/abs/S700006.html/ .
  30. J. Widengren, R. Rigler, U. Mets, “Triplet-state monitoring by fluorescence correlation spectroscopy,” J. Fluorescence 4, 255–258 (1994).
    [Crossref]

1997 (4)

K. Kneipp, Y. Wang, H. Kneipp, L. T. Perelman, I. Itzkan, R. R. Dasari, M. S. Feld, “Single molecule detection using surface-enhanced Raman scattering (SERS),” Phys. Rev. Lett. 78, 1667–1670 (1997).
[Crossref]

S. Nie, S. R. Emory, “Probing single nanoparticles by surface enhanced Raman scattering,” Science 275, 1102–1106 (1997).
[Crossref] [PubMed]

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

1996 (2)

C. Zander, M. Sauer, K. H. Drexage, D. S. Ko, A. Schultz, J. Wolfrum, L. Brand, C. Eggeling, C. A. M. Seidel, “Detection and characterization of single molecules in aqueous solution,” Appl. Phys. B 63, 517–523 (1996).

A. Spinelli, L. M. Davis, H. Dautet, “Actively quenched single-photon avalanche diode for high repetition rate time-gated photon counting,” Rev. Sci. Instrum. 67, 55–61 (1996).
[Crossref]

1995 (3)

1994 (4)

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

M. Eigen, R. Rigler, “Sorting single molecules: application to diagnostics and evolutionary biotechnology,” Proc. Natl. Acad. Sci. USA 91, 5740–5747 (1994).
[Crossref] [PubMed]

S. Nie, D. T. Chiu, R. N. Zare, “Probing individual molecules with confocal fluorescence microscopy,” Science 266, 1018–1021 (1994).
[Crossref] [PubMed]

J. Widengren, R. Rigler, U. Mets, “Triplet-state monitoring by fluorescence correlation spectroscopy,” J. Fluorescence 4, 255–258 (1994).
[Crossref]

1993 (3)

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

L. Q. Li, L. M. Davis, “Single photon avalanche diode for single molecule detection,” Rev. Sci. Instrum. 64, 1524–1529 (1993).
[Crossref]

S. A. Soper, Q. L. Mattingly, P. Vegunta, “Photon burst detection of single near-infrared fluorescent molecules,” Anal. Chem. 65, 740–747 (1993).
[Crossref]

1992 (1)

1991 (1)

1990 (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]

R. A. Mathies, K. Peck, L. Stryer, “Optimization of high-sensitivity fluorescence detection,” Anal. Chem. 62, 1786–1791 (1990).
[Crossref] [PubMed]

1976 (1)

A. Penzkofer, W. Falkenstein, W. Kaiser, “Vibronic relaxation in the S1 state of rhodamine dye solutions,” Chem. Phys. Lett. 44, 82–87 (1976).
[Crossref]

Affleck, R. L.

P. M. Goodwin, R. L. Affleck, W. P. Ambrose, J. H. Jett, M. E. Johnson, J. C. Martin, J. T. Petty, J. A. Schecker, M. Wu, R. A. Keller, “Detection of single fluorescent molecules in flowing sample streams,” in Computer Assisted Analytical Spectroscopy, S. D. Brown, ed. (Wiley, Sussex, England, 1996), pp. 61–80.

Ambrose, W. P.

P. M. Goodwin, R. L. Affleck, W. P. Ambrose, J. H. Jett, M. E. Johnson, J. C. Martin, J. T. Petty, J. A. Schecker, M. Wu, R. A. Keller, “Detection of single fluorescent molecules in flowing sample streams,” in Computer Assisted Analytical Spectroscopy, S. D. Brown, ed. (Wiley, Sussex, England, 1996), pp. 61–80.

Brand, L.

C. Zander, M. Sauer, K. H. Drexage, D. S. Ko, A. Schultz, J. Wolfrum, L. Brand, C. Eggeling, C. A. M. Seidel, “Detection and characterization of single molecules in aqueous solution,” Appl. Phys. B 63, 517–523 (1996).

Bunfield, D. B.

L. M. Davis, L. E. Schneider, D. B. Bunfield, “Increasing the rate of detection of single molecules in solution,” in Laser Applications to Chemical, Biological, and Environmental Analysis, Vol. 3 of 1996 OSA Technical Digest Series (Optical Society of America, Washington, D.C., 1996), pp. 24–26.

Bunfield, D. H.

D. H. Bunfield, “Simulation of a single molecule detection experiment,” M.S. thesis (University of Tennessee, Knoxville, Tennessee, 1997).

L. M. Davis, D. H. Bunfield, “Spectroscopic identification of individually detected fluorescent molecules,” The Fifth International Conference on Methods and Applications of Fluorescence Spectroscopy, Berlin, 1997. Book of Abstracts, W. Rettig, ed. (Springer-Verlag, Berlin, 1997), p. 27.

Castro, A.

L. M. Davis, L. Q. Li, E. B. Shera, A. Castro, S. A. Soper, “Photon statistics for the detection of single molecules in solution,” in Quantum Electronics and Laser Science, Vol. 13 of OSA Technical Digest Series (Optical Society of America (Washington, D.C., 1992), pp. 70–72.

Chiu, D. T.

S. Nie, D. T. Chiu, R. N. Zare, “Real-time detection of single molecules in solution by confocal fluorescence microscopy,” Anal. Chem. 67, 2849–2857 (1995).
[Crossref]

S. Nie, D. T. Chiu, R. N. Zare, “Probing individual molecules with confocal fluorescence microscopy,” Science 266, 1018–1021 (1994).
[Crossref] [PubMed]

Dasari, R. R.

K. Kneipp, Y. Wang, H. Kneipp, L. T. Perelman, I. Itzkan, R. R. Dasari, M. S. Feld, “Single molecule detection using surface-enhanced Raman scattering (SERS),” Phys. Rev. Lett. 78, 1667–1670 (1997).
[Crossref]

Dautet, H.

A. Spinelli, L. M. Davis, H. Dautet, “Actively quenched single-photon avalanche diode for high repetition rate time-gated photon counting,” Rev. Sci. Instrum. 67, 55–61 (1996).
[Crossref]

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 (1997).
[Crossref]

A. Spinelli, L. M. Davis, H. Dautet, “Actively quenched single-photon avalanche diode for high repetition rate time-gated photon counting,” Rev. Sci. Instrum. 67, 55–61 (1996).
[Crossref]

L. Q. Li, L. M. Davis, “Rapid and efficient detection of single chromophore molecules in aqueous solution,” Appl. Opt. 34, 3208–3217 (1995), and references cited therein.
[Crossref] [PubMed]

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

L. Q. Li, L. M. Davis, “Single photon avalanche diode for single molecule detection,” Rev. Sci. Instrum. 64, 1524–1529 (1993).
[Crossref]

S. A. Soper, L. M. Davis, E. B. Shera, “Detection and identification of single molecules in solution,” J. Opt. Soc. Am. B 9, 1761–1769 (1992).
[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]

L. M. Davis, D. H. Bunfield, “Spectroscopic identification of individually detected fluorescent molecules,” The Fifth International Conference on Methods and Applications of Fluorescence Spectroscopy, Berlin, 1997. Book of Abstracts, W. Rettig, ed. (Springer-Verlag, Berlin, 1997), p. 27.

L. M. Davis, L. Q. Li, E. B. Shera, A. Castro, S. A. Soper, “Photon statistics for the detection of single molecules in solution,” in Quantum Electronics and Laser Science, Vol. 13 of OSA Technical Digest Series (Optical Society of America (Washington, D.C., 1992), pp. 70–72.

L. M. Davis, L. Q. Li, “Monte Carlo model of a single molecule counting experiment,” in Laser Applications to Chemical Analysis, Vol. 5 of 1994 OSA Technical Digest Series (Optical Society of America, Washington, D.C., 1994), pp. 206–209.

L. M. Davis, “Efficient counting of single molecules with sub-100 μs transit times,” paper BC.06 at American Physical Society SES97 meeting, Nashville, November 1997, http://aps.org/BAPSSES97/abs/S700006.html/ .

L. M. Davis, L. E. Schneider, D. B. Bunfield, “Increasing the rate of detection of single molecules in solution,” in Laser Applications to Chemical, Biological, and Environmental Analysis, Vol. 3 of 1996 OSA Technical Digest Series (Optical Society of America, Washington, D.C., 1996), pp. 24–26.

Drexage, K. H.

C. Zander, M. Sauer, K. H. Drexage, D. S. Ko, A. Schultz, J. Wolfrum, L. Brand, C. Eggeling, C. A. M. Seidel, “Detection and characterization of single molecules in aqueous solution,” Appl. Phys. B 63, 517–523 (1996).

Eggeling, C.

C. Zander, M. Sauer, K. H. Drexage, D. S. Ko, A. Schultz, J. Wolfrum, L. Brand, C. Eggeling, C. A. M. Seidel, “Detection and characterization of single molecules in aqueous solution,” Appl. Phys. B 63, 517–523 (1996).

Eigen, M.

M. Eigen, R. Rigler, “Sorting single molecules: application to diagnostics and evolutionary biotechnology,” Proc. Natl. Acad. Sci. USA 91, 5740–5747 (1994).
[Crossref] [PubMed]

Elson, E. L.

Emory, S. R.

S. Nie, S. R. Emory, “Probing single nanoparticles by surface enhanced Raman scattering,” Science 275, 1102–1106 (1997).
[Crossref] [PubMed]

Falkenstein, W.

A. Penzkofer, W. Falkenstein, W. Kaiser, “Vibronic relaxation in the S1 state of rhodamine dye solutions,” Chem. Phys. Lett. 44, 82–87 (1976).
[Crossref]

Feld, M. S.

K. Kneipp, Y. Wang, H. Kneipp, L. T. Perelman, I. Itzkan, R. R. Dasari, M. S. Feld, “Single molecule detection using surface-enhanced Raman scattering (SERS),” Phys. Rev. Lett. 78, 1667–1670 (1997).
[Crossref]

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 (1997).
[Crossref]

Goodwin, P. M.

P. M. Goodwin, R. L. Affleck, W. P. Ambrose, J. H. Jett, M. E. Johnson, J. C. Martin, J. T. Petty, J. A. Schecker, M. Wu, R. A. Keller, “Detection of single fluorescent molecules in flowing sample streams,” in Computer Assisted Analytical Spectroscopy, S. D. Brown, ed. (Wiley, Sussex, England, 1996), pp. 61–80.

Itzkan, I.

K. Kneipp, Y. Wang, H. Kneipp, L. T. Perelman, I. Itzkan, R. R. Dasari, M. S. Feld, “Single molecule detection using surface-enhanced Raman scattering (SERS),” Phys. Rev. Lett. 78, 1667–1670 (1997).
[Crossref]

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 (1997).
[Crossref]

Jett, J. H.

P. M. Goodwin, R. L. Affleck, W. P. Ambrose, J. H. Jett, M. E. Johnson, J. C. Martin, J. T. Petty, J. A. Schecker, M. Wu, R. A. Keller, “Detection of single fluorescent molecules in flowing sample streams,” in Computer Assisted Analytical Spectroscopy, S. D. Brown, ed. (Wiley, Sussex, England, 1996), pp. 61–80.

Johnson, M. E.

P. M. Goodwin, R. L. Affleck, W. P. Ambrose, J. H. Jett, M. E. Johnson, J. C. Martin, J. T. Petty, J. A. Schecker, M. Wu, R. A. Keller, “Detection of single fluorescent molecules in flowing sample streams,” in Computer Assisted Analytical Spectroscopy, S. D. Brown, ed. (Wiley, Sussex, England, 1996), pp. 61–80.

Kaiser, W.

A. Penzkofer, W. Falkenstein, W. Kaiser, “Vibronic relaxation in the S1 state of rhodamine dye solutions,” Chem. Phys. Lett. 44, 82–87 (1976).
[Crossref]

Keller, R. A.

S. A. Soper, H. L. Nutter, R. A. Keller, L. M. Davis, E. B. Shera, “The photophysical constants of several fluorescent dyes pertaining to ultrasensitive fluorescence spectroscopy,” 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]

P. M. Goodwin, R. L. Affleck, W. P. Ambrose, J. H. Jett, M. E. Johnson, J. C. Martin, J. T. Petty, J. A. Schecker, M. Wu, R. A. Keller, “Detection of single fluorescent molecules in flowing sample streams,” in Computer Assisted Analytical Spectroscopy, S. D. Brown, ed. (Wiley, Sussex, England, 1996), pp. 61–80.

Kneipp, H.

K. Kneipp, Y. Wang, H. Kneipp, L. T. Perelman, I. Itzkan, R. R. Dasari, M. S. Feld, “Single molecule detection using surface-enhanced Raman scattering (SERS),” Phys. Rev. Lett. 78, 1667–1670 (1997).
[Crossref]

Kneipp, K.

K. Kneipp, Y. Wang, H. Kneipp, L. T. Perelman, I. Itzkan, R. R. Dasari, M. S. Feld, “Single molecule detection using surface-enhanced Raman scattering (SERS),” Phys. Rev. Lett. 78, 1667–1670 (1997).
[Crossref]

Ko, D. S.

C. Zander, M. Sauer, K. H. Drexage, D. S. Ko, A. Schultz, J. Wolfrum, L. Brand, C. Eggeling, C. A. M. Seidel, “Detection and characterization of single molecules in aqueous solution,” Appl. Phys. B 63, 517–523 (1996).

Lee, Y. H.

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

Li, L. Q.

L. Q. Li, L. M. Davis, “Rapid and efficient detection of single chromophore molecules in aqueous solution,” Appl. Opt. 34, 3208–3217 (1995), and references cited therein.
[Crossref] [PubMed]

L. Q. Li, L. M. Davis, “Single photon avalanche diode for single molecule detection,” Rev. Sci. Instrum. 64, 1524–1529 (1993).
[Crossref]

L. M. Davis, L. Q. Li, “Monte Carlo model of a single molecule counting experiment,” in Laser Applications to Chemical Analysis, Vol. 5 of 1994 OSA Technical Digest Series (Optical Society of America, Washington, D.C., 1994), pp. 206–209.

L. M. Davis, L. Q. Li, E. B. Shera, A. Castro, S. A. Soper, “Photon statistics for the detection of single molecules in solution,” in Quantum Electronics and Laser Science, Vol. 13 of OSA Technical Digest Series (Optical Society of America (Washington, D.C., 1992), pp. 70–72.

Loudon, R.

R. Loudon, The Quantum Theory of Light, 2nd ed. (Oxford University, New York, 1973), pp. 78–81.

Martin, J. C.

P. M. Goodwin, R. L. Affleck, W. P. Ambrose, J. H. Jett, M. E. Johnson, J. C. Martin, J. T. Petty, J. A. Schecker, M. Wu, R. A. Keller, “Detection of single fluorescent molecules in flowing sample streams,” in Computer Assisted Analytical Spectroscopy, S. D. Brown, ed. (Wiley, Sussex, England, 1996), pp. 61–80.

Mathies, R. A.

R. A. Mathies, K. Peck, L. Stryer, “Optimization of high-sensitivity fluorescence detection,” Anal. Chem. 62, 1786–1791 (1990).
[Crossref] [PubMed]

Mattingly, Q. L.

S. A. Soper, Q. L. Mattingly, P. Vegunta, “Photon burst detection of single near-infrared fluorescent molecules,” Anal. Chem. 65, 740–747 (1993).
[Crossref]

Maus, R. G.

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

Mertz, J.

Mets, U.

J. Widengren, R. Rigler, U. Mets, “Triplet-state monitoring by fluorescence correlation spectroscopy,” J. Fluorescence 4, 255–258 (1994).
[Crossref]

Nie, S.

S. Nie, S. R. Emory, “Probing single nanoparticles by surface enhanced Raman scattering,” Science 275, 1102–1106 (1997).
[Crossref] [PubMed]

S. Nie, D. T. Chiu, R. N. Zare, “Real-time detection of single molecules in solution by confocal fluorescence microscopy,” Anal. Chem. 67, 2849–2857 (1995).
[Crossref]

S. Nie, D. T. Chiu, R. N. Zare, “Probing individual molecules with confocal fluorescence microscopy,” Science 266, 1018–1021 (1994).
[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 fluorescent dyes pertaining to ultrasensitive fluorescence spectroscopy,” Photochem. Photobiol. 57, 972–977 (1993).
[Crossref]

Peck, K.

R. A. Mathies, K. Peck, L. Stryer, “Optimization of high-sensitivity fluorescence detection,” Anal. Chem. 62, 1786–1791 (1990).
[Crossref] [PubMed]

Penzkofer, A.

A. Penzkofer, W. Falkenstein, W. Kaiser, “Vibronic relaxation in the S1 state of rhodamine dye solutions,” Chem. Phys. Lett. 44, 82–87 (1976).
[Crossref]

Perelman, L. T.

K. Kneipp, Y. Wang, H. Kneipp, L. T. Perelman, I. Itzkan, R. R. Dasari, M. S. Feld, “Single molecule detection using surface-enhanced Raman scattering (SERS),” Phys. Rev. Lett. 78, 1667–1670 (1997).
[Crossref]

Petty, J. T.

P. M. Goodwin, R. L. Affleck, W. P. Ambrose, J. H. Jett, M. E. Johnson, J. C. Martin, J. T. Petty, J. A. Schecker, M. Wu, R. A. Keller, “Detection of single fluorescent molecules in flowing sample streams,” in Computer Assisted Analytical Spectroscopy, S. D. Brown, ed. (Wiley, Sussex, England, 1996), pp. 61–80.

Qian, H.

Ramsey, J. 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 (1997).
[Crossref]

Rigler, R.

M. Eigen, R. Rigler, “Sorting single molecules: application to diagnostics and evolutionary biotechnology,” Proc. Natl. Acad. Sci. USA 91, 5740–5747 (1994).
[Crossref] [PubMed]

J. Widengren, R. Rigler, U. Mets, “Triplet-state monitoring by fluorescence correlation spectroscopy,” J. Fluorescence 4, 255–258 (1994).
[Crossref]

Sauer, M.

C. Zander, M. Sauer, K. H. Drexage, D. S. Ko, A. Schultz, J. Wolfrum, L. Brand, C. Eggeling, C. A. M. Seidel, “Detection and characterization of single molecules in aqueous solution,” Appl. Phys. B 63, 517–523 (1996).

Schecker, J. A.

P. M. Goodwin, R. L. Affleck, W. P. Ambrose, J. H. Jett, M. E. Johnson, J. C. Martin, J. T. Petty, J. A. Schecker, M. Wu, R. A. Keller, “Detection of single fluorescent molecules in flowing sample streams,” in Computer Assisted Analytical Spectroscopy, S. D. Brown, ed. (Wiley, Sussex, England, 1996), pp. 61–80.

Schneider, L. E.

L. M. Davis, L. E. Schneider, D. B. Bunfield, “Increasing the rate of detection of single molecules in solution,” in Laser Applications to Chemical, Biological, and Environmental Analysis, Vol. 3 of 1996 OSA Technical Digest Series (Optical Society of America, Washington, D.C., 1996), pp. 24–26.

Schultz, A.

C. Zander, M. Sauer, K. H. Drexage, D. S. Ko, A. Schultz, J. Wolfrum, L. Brand, C. Eggeling, C. A. M. Seidel, “Detection and characterization of single molecules in aqueous solution,” Appl. Phys. B 63, 517–523 (1996).

Seidel, C. A. M.

C. Zander, M. Sauer, K. H. Drexage, D. S. Ko, A. Schultz, J. Wolfrum, L. Brand, C. Eggeling, C. A. M. Seidel, “Detection and characterization of single molecules in aqueous solution,” Appl. Phys. B 63, 517–523 (1996).

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 fluorescent dyes pertaining to ultrasensitive fluorescence spectroscopy,” Photochem. Photobiol. 57, 972–977 (1993).
[Crossref]

S. A. Soper, L. M. Davis, E. B. Shera, “Detection and identification of single molecules in solution,” J. Opt. Soc. Am. B 9, 1761–1769 (1992).
[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]

L. M. Davis, L. Q. Li, E. B. Shera, A. Castro, S. A. Soper, “Photon statistics for the detection of single molecules in solution,” in Quantum Electronics and Laser Science, Vol. 13 of OSA Technical Digest Series (Optical Society of America (Washington, D.C., 1992), pp. 70–72.

Smith, B. W.

Y. H. Lee, R. G. Maus, B. W. 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, Q. L. Mattingly, P. Vegunta, “Photon burst detection of single near-infrared fluorescent molecules,” Anal. Chem. 65, 740–747 (1993).
[Crossref]

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

S. A. Soper, L. M. Davis, E. B. Shera, “Detection and identification of single molecules in solution,” J. Opt. Soc. Am. B 9, 1761–1769 (1992).
[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]

L. M. Davis, L. Q. Li, E. B. Shera, A. Castro, S. A. Soper, “Photon statistics for the detection of single molecules in solution,” in Quantum Electronics and Laser Science, Vol. 13 of OSA Technical Digest Series (Optical Society of America (Washington, D.C., 1992), pp. 70–72.

Spinelli, A.

A. Spinelli, L. M. Davis, H. Dautet, “Actively quenched single-photon avalanche diode for high repetition rate time-gated photon counting,” Rev. Sci. Instrum. 67, 55–61 (1996).
[Crossref]

Stryer, L.

R. A. Mathies, K. Peck, L. Stryer, “Optimization of high-sensitivity fluorescence detection,” Anal. Chem. 62, 1786–1791 (1990).
[Crossref] [PubMed]

Vegunta, P.

S. A. Soper, Q. L. Mattingly, P. Vegunta, “Photon burst detection of single near-infrared fluorescent molecules,” Anal. Chem. 65, 740–747 (1993).
[Crossref]

Wang, Y.

K. Kneipp, Y. Wang, H. Kneipp, L. T. Perelman, I. Itzkan, R. R. Dasari, M. S. Feld, “Single molecule detection using surface-enhanced Raman scattering (SERS),” Phys. Rev. Lett. 78, 1667–1670 (1997).
[Crossref]

Webb, W. W.

Widengren, J.

J. Widengren, R. Rigler, U. Mets, “Triplet-state monitoring by fluorescence correlation spectroscopy,” J. Fluorescence 4, 255–258 (1994).
[Crossref]

Winefordner, J. D.

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

Wolfrum, J.

C. Zander, M. Sauer, K. H. Drexage, D. S. Ko, A. Schultz, J. Wolfrum, L. Brand, C. Eggeling, C. A. M. Seidel, “Detection and characterization of single molecules in aqueous solution,” Appl. Phys. B 63, 517–523 (1996).

Wu, M.

P. M. Goodwin, R. L. Affleck, W. P. Ambrose, J. H. Jett, M. E. Johnson, J. C. Martin, J. T. Petty, J. A. Schecker, M. Wu, R. A. Keller, “Detection of single fluorescent molecules in flowing sample streams,” in Computer Assisted Analytical Spectroscopy, S. D. Brown, ed. (Wiley, Sussex, England, 1996), pp. 61–80.

Xu, C.

Xu, X. H.

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

Zander, C.

C. Zander, M. Sauer, K. H. Drexage, D. S. Ko, A. Schultz, J. Wolfrum, L. Brand, C. Eggeling, C. A. M. Seidel, “Detection and characterization of single molecules in aqueous solution,” Appl. Phys. B 63, 517–523 (1996).

Zare, R. N.

S. Nie, D. T. Chiu, R. N. Zare, “Real-time detection of single molecules in solution by confocal fluorescence microscopy,” Anal. Chem. 67, 2849–2857 (1995).
[Crossref]

S. Nie, D. T. Chiu, R. N. Zare, “Probing individual molecules with confocal fluorescence microscopy,” Science 266, 1018–1021 (1994).
[Crossref] [PubMed]

Anal. Chem. (5)

S. A. Soper, Q. L. Mattingly, P. Vegunta, “Photon burst detection of single near-infrared fluorescent molecules,” Anal. Chem. 65, 740–747 (1993).
[Crossref]

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

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 (1997).
[Crossref]

S. Nie, D. T. Chiu, R. N. Zare, “Real-time detection of single molecules in solution by confocal fluorescence microscopy,” Anal. Chem. 67, 2849–2857 (1995).
[Crossref]

R. A. Mathies, K. Peck, L. Stryer, “Optimization of high-sensitivity fluorescence detection,” Anal. Chem. 62, 1786–1791 (1990).
[Crossref] [PubMed]

Appl. Opt. (2)

Appl. Phys. B (1)

C. Zander, M. Sauer, K. H. Drexage, D. S. Ko, A. Schultz, J. Wolfrum, L. Brand, C. Eggeling, C. A. M. Seidel, “Detection and characterization of single molecules in aqueous solution,” Appl. Phys. B 63, 517–523 (1996).

Chem. Phys. Lett. (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]

A. Penzkofer, W. Falkenstein, W. Kaiser, “Vibronic relaxation in the S1 state of rhodamine dye solutions,” Chem. Phys. Lett. 44, 82–87 (1976).
[Crossref]

J. Fluorescence (1)

J. Widengren, R. Rigler, U. Mets, “Triplet-state monitoring by fluorescence correlation spectroscopy,” J. Fluorescence 4, 255–258 (1994).
[Crossref]

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

Opt. Lett. (1)

Photochem. Photobiol. (1)

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

Phys. Rev. Lett. (1)

K. Kneipp, Y. Wang, H. Kneipp, L. T. Perelman, I. Itzkan, R. R. Dasari, M. S. Feld, “Single molecule detection using surface-enhanced Raman scattering (SERS),” Phys. Rev. Lett. 78, 1667–1670 (1997).
[Crossref]

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

M. Eigen, R. Rigler, “Sorting single molecules: application to diagnostics and evolutionary biotechnology,” Proc. Natl. Acad. Sci. USA 91, 5740–5747 (1994).
[Crossref] [PubMed]

Rev. Sci. Instrum. (2)

L. Q. Li, L. M. Davis, “Single photon avalanche diode for single molecule detection,” Rev. Sci. Instrum. 64, 1524–1529 (1993).
[Crossref]

A. Spinelli, L. M. Davis, H. Dautet, “Actively quenched single-photon avalanche diode for high repetition rate time-gated photon counting,” Rev. Sci. Instrum. 67, 55–61 (1996).
[Crossref]

Science (3)

S. Nie, D. T. Chiu, R. N. Zare, “Probing individual molecules with confocal fluorescence microscopy,” Science 266, 1018–1021 (1994).
[Crossref] [PubMed]

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

S. Nie, S. R. Emory, “Probing single nanoparticles by surface enhanced Raman scattering,” Science 275, 1102–1106 (1997).
[Crossref] [PubMed]

Other (9)

D. H. Bunfield, “Simulation of a single molecule detection experiment,” M.S. thesis (University of Tennessee, Knoxville, Tennessee, 1997).

L. M. Davis, D. H. Bunfield, “Spectroscopic identification of individually detected fluorescent molecules,” The Fifth International Conference on Methods and Applications of Fluorescence Spectroscopy, Berlin, 1997. Book of Abstracts, W. Rettig, ed. (Springer-Verlag, Berlin, 1997), p. 27.

One significant application currently under development that demands efficient and unambiguous SMD is rapid DNA sequencing. Also, efficient processing of the sample is desirable for assay of miniscule sample quantities, such as the components of a single cell and genetic screening without DNA amplification by polymerase chain reaction. See references of Ref. 1.

R. Loudon, The Quantum Theory of Light, 2nd ed. (Oxford University, New York, 1973), pp. 78–81.

P. M. Goodwin, R. L. Affleck, W. P. Ambrose, J. H. Jett, M. E. Johnson, J. C. Martin, J. T. Petty, J. A. Schecker, M. Wu, R. A. Keller, “Detection of single fluorescent molecules in flowing sample streams,” in Computer Assisted Analytical Spectroscopy, S. D. Brown, ed. (Wiley, Sussex, England, 1996), pp. 61–80.

L. M. Davis, L. Q. Li, “Monte Carlo model of a single molecule counting experiment,” in Laser Applications to Chemical Analysis, Vol. 5 of 1994 OSA Technical Digest Series (Optical Society of America, Washington, D.C., 1994), pp. 206–209.

L. M. Davis, L. E. Schneider, D. B. Bunfield, “Increasing the rate of detection of single molecules in solution,” in Laser Applications to Chemical, Biological, and Environmental Analysis, Vol. 3 of 1996 OSA Technical Digest Series (Optical Society of America, Washington, D.C., 1996), pp. 24–26.

L. M. Davis, “Efficient counting of single molecules with sub-100 μs transit times,” paper BC.06 at American Physical Society SES97 meeting, Nashville, November 1997, http://aps.org/BAPSSES97/abs/S700006.html/ .

L. M. Davis, L. Q. Li, E. B. Shera, A. Castro, S. A. Soper, “Photon statistics for the detection of single molecules in solution,” in Quantum Electronics and Laser Science, Vol. 13 of OSA Technical Digest Series (Optical Society of America (Washington, D.C., 1992), pp. 70–72.

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Figures (6)

Fig. 1
Fig. 1

Schematic of flow cell and experimental setup. A beam of picosecond pulses from a synchronously pumped dye laser is focused into an 800-μm square-bore capillary. Fluorescence from molecules transiting the beam is collected at right angles, spatially filtered, spectrally filtered by an interference filter, and focused onto a SPAD. Raman scatter from the solvent that overlaps the fluorescence band is discriminated with use of a custom-built subnanosecond time-gating circuit and also a time-to-amplitude converter, fast digitizer, and software filter. The digitized data stream is further filtered to identify individual molecule transits. See Ref. 1 for further details.

Fig. 2
Fig. 2

Determination of the efficiency of light collection through the spatial filter.

Fig. 3
Fig. 3

Flow chart of overall simulation. All stochastic steps are indicated in italics.

Fig. 4
Fig. 4

Results of simulation under conditions of Table 1. (a) Geometry of probe region, showing focused laser beam, region of maximum collection efficiency (wire grid), and trajectories of three molecules, with darker segments indicating that the molecule is in the triplet manifold; (b) individual photons and output from weighted sliding sum (wss) during transit of molecule 6; (c) output from wss during passage of ten molecules, with histogram of peak amplitudes from 10,000 molecules drawn against the y ordinate; (d) autocorrelation function and Gaussian curve fit.

Fig. 5
Fig. 5

Histogram of peak amplitudes under fast detection conditions.

Fig. 6
Fig. 6

Autocorrelation function under fast detection conditions.

Tables (2)

Tables Icon

Table 1 Parameters Used to Simulate Experiment of Ref. 1

Tables Icon

Table 2 Changes in Parameters for Rapid Detection

Equations (40)

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

Δ z = ν Δ t .
t 2 = R EX δ t ,
δ t = π r 0 2 M N A Δ z - 1 ,
c = 4 π Dt - 3 / 2 exp - x 2 + y 2 + z 2 / 4 Dt .
ω ¯ i 0 = M i ω i 0 ,   i = y , z .
Î = 2 P / π ω ¯ y 0 ω ¯ z 0 ,
I x ,   y ,   z = i = y , z ω ¯ i 0 ω ¯ i x exp - 2 i 2 ω ¯ i 2 x ,
ω ¯ i 2 x = ω ¯ i 0 2 1 + x - x i / x i 0 2 ,
x ¯ i 0 = π ω ¯ i 0 2 / M i 2 λ = x i 0 ,
K = σ a ζ Î I x ,   y ,   z / E γ ,
d d t P 0 P 1 P 2 = - K K τ f - 1 K - K + τ v - 1 0 0 τ ν - 1 τ f - 1 P 0 P 1 P 2 ,
P ˆ = P ¯ / 2 π   R σ t ,
P t = P ˆ   exp - t 2 / 2 σ t 2 .
K t = K ˆ   exp - t 2 / 2 σ t 2 ,
K ˆ = 2 σ a ζ t P ˆ I x ,   y ,   z / π ω ¯ y 0 ω ¯ z 0 E γ .
P E =   K t d t ,
  K t d t = 2 σ a ζ P ¯ I x ,   y ,   z / π ω ¯ y 0 ω ¯ z 0 E γ R .
P E = 1 - exp   K t d t .
P E = 1 - exp   K t d t / 2 .
P f = P E Φ f Δ t / R ,
P isc = P E Φ isc Δ t / R ,
P d = P E Φ d Δ t / R ,
Ĉ = 1 - cos   α / 2 ,
α = sin - 1 NA / n ,
A 1 ,   Z ,   R = β + γ Z 2 - sin   2 β + Z   sin   2 γ / 2 ,
β = cos - 1 1 + R 2 - Z 2 / 2 R ,
γ = cos - 1 Z + R 2 - 1 2 / 2 RZ .
C R , Z = 1 , 0 < Z < 1 - R A 1 ,   Z ,   R / π Z 2 , 1 - R < Z < 1 + R 1 - Z / tan 2   α + Z 2 / 2 π Ĉ 1 + R < Z < R - 1 0 , R - 1 < Z ,
R = y   tan   α / s 0 ,
R = x 2 + z 2 / s 0 ,
V = C x , y , z I x , y , z d x d y d z
b b = β β VP Ĉ / ω ¯ y 0 ω ¯ z 0 ,
β β = b ¯ b ¯ ω ¯ ¯ y 0 ω ¯ ¯ z 0 / V ¯ P ¯ Ĉ ¯ ,
G m = n   s n s n + m .
s ¯ n     exp - 2 n 2 / δ t 2 ,
δ t = ω ¯ z 0 / Δ z
w k = 2 exp - 2 k 2 / δ t 2 ,   k = - q , , q ,
S n = i = - q k = q   s n + k w k .
Ŝ = k = - q k = 1   w k N 2 / 2 π   δ t exp - 2 k 2 / δ t 2 0.95 N .
S n = - q < t 1 p - n < q   w t 1 p - n ,

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