Abstract

Recent developments in laser-based detection of fluorescent molecules have made possible the implementation of very sensitive techniques for biochemical analysis. We present and discuss our experiments on the applications of our recently developed technique of single-molecule detection to the analysis of molecules of biological interest. These newly developed methods are capable of detecting and identifying biomolecules at the single-molecule level of sensitivity. In one case, identification is based on measuring fluorescence brightness from single molecules. In another, molecules are classified by determining their electrophoretic velocities.

© 1995 Optical Society of America

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  1. 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]
  2. 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]
  3. T. Hirshfield, “Optical microscopic observation of single small molecules,” Appl. Opt. 15, 2965–2966 (1976).
    [Crossref]
  4. K. Peck, L. Stryer, A. N. Glazer, R. A. Mathies, “Single-molecule fluorescence detection: autocorrelation criterion and experimental realization with phycoerythrin,” Proc. Natl. Acad. Sci. USA 86, 4087–4091 (1989).
    [Crossref] [PubMed]
  5. R. P. Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Eugene, Or., 1992), p. 77.
  6. 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]
  7. M. Eigen, R. Rigler, “Sorting single molecules: application to diagnostics and evolutionary biotechnology,” Proc. Natl. Acad. Sci. USA 91, 5740–5747 (1994).
    [Crossref] [PubMed]
  8. S. A. Soper, Q. L. Mattingly, P. Vegunta, “Photon burst detection of single near infrared fluorescent molecules,” Anal. Chem. 65, 740–747 (1993).
    [Crossref]
  9. A. Castro, F. R. Fairfield, E. B. Shera, “Fluorescence detection and size measurement of single DNA molecules,” Anal. Chem. 65, 849–852 (1993).
    [Crossref]
  10. D. Pinkel, R. Stovel, “Flow chambers and sample handling,” in Flow Cytometry: Instrumentation and Data Analysis, M. A. Van Dilla, P. N. Dean, O. D. Laerum, M. R. Melamed, eds. (Academic, London, 1985), Chap. 3.
  11. Molecular Probes, Inc., Eugene, Or.
  12. Z. Huang, Molecular Probes, Inc., 4849 Pitchford Ave., Eugene, Or., 97402 (personal communication, 1992).
  13. H. S. Rye, S. Yue, D. E. Wemmer, M. A. Quesada, R. P. Haugland, R. A. Mathies, A. N. Glazer, “Stable fluorescent complexes of double-stranded DNAwith bisintercalating asymmetric cyanine dyes—properties and applications,” Nucleic Acids Res. 20, 2803–2812 (1992).
    [Crossref] [PubMed]
  14. B. Yoshinobu, C. Sumita, K. Hide, N. Ishimaru, K. Samata, A. Tanaka, M. Tsuhako, “Separation of DNA fragments by high-performance liquid chromatography and capillary electrophoresis,” J. Liq. Chromatogr. 16, 955–965 (1993).
    [Crossref]
  15. M. Zhu, D. L. Hansen, S. Burd, F. Gannon, “Factors affecting free zone electrophoresis and isoelectric focusing in capillary electrophoresis,” J. Chromatogr. 480, 311–319 (1989).
    [Crossref]
  16. C. Tanford, Physical Chemistry of Macromolecules (Wiley, New York, 1961), p. 361.
  17. A. K. Bej, M. H. Mahbubani, R. M. Atlas, “Amplification of nucleic acids by polymerase chain reaction (PCR) and other methods and their applications,” Crit. Rev. Biochem. Biophys. 26, 301–334 (1991).
    [Crossref]

1994 (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]

1993 (4)

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

A. Castro, F. R. Fairfield, E. B. Shera, “Fluorescence detection and size measurement of single DNA molecules,” Anal. Chem. 65, 849–852 (1993).
[Crossref]

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]

B. Yoshinobu, C. Sumita, K. Hide, N. Ishimaru, K. Samata, A. Tanaka, M. Tsuhako, “Separation of DNA fragments by high-performance liquid chromatography and capillary electrophoresis,” J. Liq. Chromatogr. 16, 955–965 (1993).
[Crossref]

1992 (2)

H. S. Rye, S. Yue, D. E. Wemmer, M. A. Quesada, R. P. Haugland, R. A. Mathies, A. N. Glazer, “Stable fluorescent complexes of double-stranded DNAwith bisintercalating asymmetric cyanine dyes—properties and applications,” Nucleic Acids Res. 20, 2803–2812 (1992).
[Crossref] [PubMed]

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]

1991 (1)

A. K. Bej, M. H. Mahbubani, R. M. Atlas, “Amplification of nucleic acids by polymerase chain reaction (PCR) and other methods and their applications,” Crit. Rev. Biochem. Biophys. 26, 301–334 (1991).
[Crossref]

1990 (1)

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]

1989 (2)

M. Zhu, D. L. Hansen, S. Burd, F. Gannon, “Factors affecting free zone electrophoresis and isoelectric focusing in capillary electrophoresis,” J. Chromatogr. 480, 311–319 (1989).
[Crossref]

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

1976 (1)

Atlas, R. M.

A. K. Bej, M. H. Mahbubani, R. M. Atlas, “Amplification of nucleic acids by polymerase chain reaction (PCR) and other methods and their applications,” Crit. Rev. Biochem. Biophys. 26, 301–334 (1991).
[Crossref]

Barnes, M. D.

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]

Bej, A. K.

A. K. Bej, M. H. Mahbubani, R. M. Atlas, “Amplification of nucleic acids by polymerase chain reaction (PCR) and other methods and their applications,” Crit. Rev. Biochem. Biophys. 26, 301–334 (1991).
[Crossref]

Burd, S.

M. Zhu, D. L. Hansen, S. Burd, F. Gannon, “Factors affecting free zone electrophoresis and isoelectric focusing in capillary electrophoresis,” J. Chromatogr. 480, 311–319 (1989).
[Crossref]

Castro, A.

A. Castro, F. R. Fairfield, E. B. Shera, “Fluorescence detection and size measurement of single DNA molecules,” Anal. Chem. 65, 849–852 (1993).
[Crossref]

Davis, L. M.

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]

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]

Fairfield, F. R.

A. Castro, F. R. Fairfield, E. B. Shera, “Fluorescence detection and size measurement of single DNA molecules,” Anal. Chem. 65, 849–852 (1993).
[Crossref]

Gannon, F.

M. Zhu, D. L. Hansen, S. Burd, F. Gannon, “Factors affecting free zone electrophoresis and isoelectric focusing in capillary electrophoresis,” J. Chromatogr. 480, 311–319 (1989).
[Crossref]

Glazer, A. N.

H. S. Rye, S. Yue, D. E. Wemmer, M. A. Quesada, R. P. Haugland, R. A. Mathies, A. N. Glazer, “Stable fluorescent complexes of double-stranded DNAwith bisintercalating asymmetric cyanine dyes—properties and applications,” Nucleic Acids Res. 20, 2803–2812 (1992).
[Crossref] [PubMed]

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

Hansen, D. L.

M. Zhu, D. L. Hansen, S. Burd, F. Gannon, “Factors affecting free zone electrophoresis and isoelectric focusing in capillary electrophoresis,” J. Chromatogr. 480, 311–319 (1989).
[Crossref]

Haugland, R. P.

H. S. Rye, S. Yue, D. E. Wemmer, M. A. Quesada, R. P. Haugland, R. A. Mathies, A. N. Glazer, “Stable fluorescent complexes of double-stranded DNAwith bisintercalating asymmetric cyanine dyes—properties and applications,” Nucleic Acids Res. 20, 2803–2812 (1992).
[Crossref] [PubMed]

R. P. Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Eugene, Or., 1992), p. 77.

Hide, K.

B. Yoshinobu, C. Sumita, K. Hide, N. Ishimaru, K. Samata, A. Tanaka, M. Tsuhako, “Separation of DNA fragments by high-performance liquid chromatography and capillary electrophoresis,” J. Liq. Chromatogr. 16, 955–965 (1993).
[Crossref]

Hirshfield, T.

Huang, Z.

Z. Huang, Molecular Probes, Inc., 4849 Pitchford Ave., Eugene, Or., 97402 (personal communication, 1992).

Ishimaru, N.

B. Yoshinobu, C. Sumita, K. Hide, N. Ishimaru, K. Samata, A. Tanaka, M. Tsuhako, “Separation of DNA fragments by high-performance liquid chromatography and capillary electrophoresis,” J. Liq. Chromatogr. 16, 955–965 (1993).
[Crossref]

Keller, R. A.

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]

Mahbubani, M. H.

A. K. Bej, M. H. Mahbubani, R. M. Atlas, “Amplification of nucleic acids by polymerase chain reaction (PCR) and other methods and their applications,” Crit. Rev. Biochem. Biophys. 26, 301–334 (1991).
[Crossref]

Mathies, R. A.

H. S. Rye, S. Yue, D. E. Wemmer, M. A. Quesada, R. P. Haugland, R. A. Mathies, A. N. Glazer, “Stable fluorescent complexes of double-stranded DNAwith bisintercalating asymmetric cyanine dyes—properties and applications,” Nucleic Acids Res. 20, 2803–2812 (1992).
[Crossref] [PubMed]

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

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]

Peck, K.

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

Pinkel, D.

D. Pinkel, R. Stovel, “Flow chambers and sample handling,” in Flow Cytometry: Instrumentation and Data Analysis, M. A. Van Dilla, P. N. Dean, O. D. Laerum, M. R. Melamed, eds. (Academic, London, 1985), Chap. 3.

Quesada, M. A.

H. S. Rye, S. Yue, D. E. Wemmer, M. A. Quesada, R. P. Haugland, R. A. Mathies, A. N. Glazer, “Stable fluorescent complexes of double-stranded DNAwith bisintercalating asymmetric cyanine dyes—properties and applications,” Nucleic Acids Res. 20, 2803–2812 (1992).
[Crossref] [PubMed]

Ramsey, J. M.

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.

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

Rye, H. S.

H. S. Rye, S. Yue, D. E. Wemmer, M. A. Quesada, R. P. Haugland, R. A. Mathies, A. N. Glazer, “Stable fluorescent complexes of double-stranded DNAwith bisintercalating asymmetric cyanine dyes—properties and applications,” Nucleic Acids Res. 20, 2803–2812 (1992).
[Crossref] [PubMed]

Samata, K.

B. Yoshinobu, C. Sumita, K. Hide, N. Ishimaru, K. Samata, A. Tanaka, M. Tsuhako, “Separation of DNA fragments by high-performance liquid chromatography and capillary electrophoresis,” J. Liq. Chromatogr. 16, 955–965 (1993).
[Crossref]

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.

A. Castro, F. R. Fairfield, E. B. Shera, “Fluorescence detection and size measurement of single DNA molecules,” Anal. Chem. 65, 849–852 (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]

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, 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]

Stovel, R.

D. Pinkel, R. Stovel, “Flow chambers and sample handling,” in Flow Cytometry: Instrumentation and Data Analysis, M. A. Van Dilla, P. N. Dean, O. D. Laerum, M. R. Melamed, eds. (Academic, London, 1985), Chap. 3.

Stryer, L.

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

Sumita, C.

B. Yoshinobu, C. Sumita, K. Hide, N. Ishimaru, K. Samata, A. Tanaka, M. Tsuhako, “Separation of DNA fragments by high-performance liquid chromatography and capillary electrophoresis,” J. Liq. Chromatogr. 16, 955–965 (1993).
[Crossref]

Tanaka, A.

B. Yoshinobu, C. Sumita, K. Hide, N. Ishimaru, K. Samata, A. Tanaka, M. Tsuhako, “Separation of DNA fragments by high-performance liquid chromatography and capillary electrophoresis,” J. Liq. Chromatogr. 16, 955–965 (1993).
[Crossref]

Tanford, C.

C. Tanford, Physical Chemistry of Macromolecules (Wiley, New York, 1961), p. 361.

Tsuhako, M.

B. Yoshinobu, C. Sumita, K. Hide, N. Ishimaru, K. Samata, A. Tanaka, M. Tsuhako, “Separation of DNA fragments by high-performance liquid chromatography and capillary electrophoresis,” J. Liq. Chromatogr. 16, 955–965 (1993).
[Crossref]

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]

Wemmer, D. E.

H. S. Rye, S. Yue, D. E. Wemmer, M. A. Quesada, R. P. Haugland, R. A. Mathies, A. N. Glazer, “Stable fluorescent complexes of double-stranded DNAwith bisintercalating asymmetric cyanine dyes—properties and applications,” Nucleic Acids Res. 20, 2803–2812 (1992).
[Crossref] [PubMed]

Whitten, W. B.

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]

Yoshinobu, B.

B. Yoshinobu, C. Sumita, K. Hide, N. Ishimaru, K. Samata, A. Tanaka, M. Tsuhako, “Separation of DNA fragments by high-performance liquid chromatography and capillary electrophoresis,” J. Liq. Chromatogr. 16, 955–965 (1993).
[Crossref]

Yue, S.

H. S. Rye, S. Yue, D. E. Wemmer, M. A. Quesada, R. P. Haugland, R. A. Mathies, A. N. Glazer, “Stable fluorescent complexes of double-stranded DNAwith bisintercalating asymmetric cyanine dyes—properties and applications,” Nucleic Acids Res. 20, 2803–2812 (1992).
[Crossref] [PubMed]

Zhu, M.

M. Zhu, D. L. Hansen, S. Burd, F. Gannon, “Factors affecting free zone electrophoresis and isoelectric focusing in capillary electrophoresis,” J. Chromatogr. 480, 311–319 (1989).
[Crossref]

Anal. Chem. (3)

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

A. Castro, F. R. Fairfield, E. B. Shera, “Fluorescence detection and size measurement of single DNA molecules,” Anal. Chem. 65, 849–852 (1993).
[Crossref]

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]

Appl. Opt. (1)

Chem. Phys. Lett. (1)

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]

Crit. Rev. Biochem. Biophys. (1)

A. K. Bej, M. H. Mahbubani, R. M. Atlas, “Amplification of nucleic acids by polymerase chain reaction (PCR) and other methods and their applications,” Crit. Rev. Biochem. Biophys. 26, 301–334 (1991).
[Crossref]

J. Chromatogr. (1)

M. Zhu, D. L. Hansen, S. Burd, F. Gannon, “Factors affecting free zone electrophoresis and isoelectric focusing in capillary electrophoresis,” J. Chromatogr. 480, 311–319 (1989).
[Crossref]

J. Liq. Chromatogr. (1)

B. Yoshinobu, C. Sumita, K. Hide, N. Ishimaru, K. Samata, A. Tanaka, M. Tsuhako, “Separation of DNA fragments by high-performance liquid chromatography and capillary electrophoresis,” J. Liq. Chromatogr. 16, 955–965 (1993).
[Crossref]

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

Nucleic Acids Res. (1)

H. S. Rye, S. Yue, D. E. Wemmer, M. A. Quesada, R. P. Haugland, R. A. Mathies, A. N. Glazer, “Stable fluorescent complexes of double-stranded DNAwith bisintercalating asymmetric cyanine dyes—properties and applications,” Nucleic Acids Res. 20, 2803–2812 (1992).
[Crossref] [PubMed]

Proc. Natl. Acad. Sci. USA (2)

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

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

Other (5)

R. P. Haugland, Handbook of Fluorescent Probes and Research Chemicals (Molecular Probes, Eugene, Or., 1992), p. 77.

D. Pinkel, R. Stovel, “Flow chambers and sample handling,” in Flow Cytometry: Instrumentation and Data Analysis, M. A. Van Dilla, P. N. Dean, O. D. Laerum, M. R. Melamed, eds. (Academic, London, 1985), Chap. 3.

Molecular Probes, Inc., Eugene, Or.

Z. Huang, Molecular Probes, Inc., 4849 Pitchford Ave., Eugene, Or., 97402 (personal communication, 1992).

C. Tanford, Physical Chemistry of Macromolecules (Wiley, New York, 1961), p. 361.

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Figures (5)

Fig. 1
Fig. 1

Weighted-sum plots for (top) a 10 fM-rhodamine 6G aqueous solution and (bottom) neat water. Laser power, 5 mW; flow velocity, 200 μm/s.

Fig. 2
Fig. 2

Histogram of single-fragment fluorescence intensities for a mixture of λ DNA (48 Kbp) and λ DNA Xba I digest (24 Kbp) at 3 × 10−15 M each.

Fig. 3
Fig. 3

Schematic drawing of the experimental setup for SME electrophoresis.

Fig. 4
Fig. 4

Single-fragment fluorescence bursts for a 10-fM solution of λ DNA in a TBE buffer.

Fig. 5
Fig. 5

Histogram of individual electrophoretic velocities for a mixture of 38-, 24-, and 10-Kbp DNA fragments in TBE/hydroxy-propyl-methyl-cellulose solution (10 fM each).

Equations (1)

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S ( t ) = τ = 0 k - 1 ω ( τ ) d ( t + τ ) ,

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