Abstract

We characterize a new geometry for single-molecule detection with flow for use with a submilliliter drop of sample on an inverted confocal microscope. The solution is sucked into a glass capillary positioned above the ellipsoidal confocal volume so that molecules traverse the longest axis of the ellipsoid for greatest photon yield. Decreased spacing between the capillary tip and laser focus gives increased flow speed, as measured by fluorescence correlation spectroscopy, but also increased background from capillary autofluorescence. Flow can alleviate localized triplet and photobleaching effects and speed single-molecule sampling rates for fluorescence fluctuation spectroscopy determinations of slowly diffusing biomolecules in pharmaceutical drug discovery research.

© 2007 Optical Society of America

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    [CrossRef] [PubMed]
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    [CrossRef] [PubMed]
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    [CrossRef]
  46. M. A. Holden, S. Kumar, E. T. Castellana, A. Beskok, and P. S. Cremer, "Generating fixed concentration arrays in a microfluidic device," Sens. Actuators B 92, 199-207 (2003).
    [CrossRef]

2005 (6)

L. A. A. de Jong, D. R. A. Uges, J. P. Franke, and R. Bischoff, "Receptor-ligand binding assays: technologies and applications," J. Chromatogr. B: Biomed Sci. Appl. 829, 1-25 (2005).
[CrossRef]

S. M. Stavis, J. B. Edel, K. T. Samiee, and H. G. Craighead, "Single molecule studies of quantum dot conjugates in a submicrometer fluidic channel," Lab Chip 5, 337-343 (2005).
[CrossRef] [PubMed]

P. S. Dittrich and A. Manz, "Single-molecule fluorescence detection in microfluidic channels--the Holy Grail in μTAS," Anal. Bioanal. Chem. 382, 1771-1782 (2005).
[CrossRef] [PubMed]

J. Melin, H. Johansson, O. Soderberg, F. Nikolajeff, U. Landegren, M. Nilsson, and J. Jarvius, "Thermoplastic microfluidic platform for single-molecule detection, cell culture, and actuation," Anal. Chem. 77, 7122-7130 (2005).
[CrossRef] [PubMed]

A. J. Skulan, L. M. Barrett, A. K. Singh, E. B. Cummings, and G. J. Flechtner, "Fabrication and analysis of spatially uniform field electrokinetic flow devices: theory and experiment," Anal. Chem. 77, 6790-6797 (2005).
[CrossRef] [PubMed]

L. M. Davis, G. Q. Shen, and D. A. Ball, "Saturation effects in fluorescence correlation spectroscopy," in Multiphoton Microscopy in the Biomedical Sciences, A. Periasamy, P. T. C. So, eds., Proc. SPIE 5700, 128-137 (2005).
[CrossRef]

2004 (3)

B. H. Kunst, A. Schots, and A. J. W. G. Visser, "Design of a confocal microfluidic particle sorter using fluorescent photon burst detection," Rev. Sci. Instrum. 75, 2892-2898 (2004).
[CrossRef]

M. E. Johnson and J. P. Landers, "Fundamentals and practice for ultrasensitive laser-induced fluorescence detection in microanalytical systems," Electrophoresis 25, 3513-3527 (2004).
[CrossRef] [PubMed]

K. O. Greulich, "Single molecule techniques for biomedicine and pharmacology," Curr. Pharm. Biotechnol. 5, 243-259 (2004).
[CrossRef] [PubMed]

2003 (6)

J. P. Shelby and D. T. Chiu, "Mapping fast flows over micrometer-length scales using flow-tagging velocimetry and single-molecule detection," Anal. Chem. 75, 1387-1392 (2003).
[CrossRef] [PubMed]

P. S. Dittrich and P. Schwille, "An integrated microfluidic system for reaction, high-sensitivity detection, and sorting of fluorescent cells and particles," Anal. Chem. 75, 5767-5774 (2003).
[CrossRef] [PubMed]

L. M. Davis, P. E. Williams, D. A. Ball, E. D. Matayoshi, and K. M. Swift, "Data reduction methods for application of fluorescence correlation spectroscopy to pharmaceutical drug discovery," Curr. Pharm. Biotechnol. 4, 451-462 (2003).
[CrossRef] [PubMed]

J. J. Zheng and E. S. Yeung, "Counting single DNA molecules in a capillary with radial focusing," Aust. J. Chem. 56, 149-153 (2003).
[CrossRef]

A. Lundqvist, D. T. Chiu, and O. Orwar, "Electrophoretic separation and confocal laser-induced fluorescence detection at ultralow concentrations in constricted fused-silica capillaries," Electrophoresis 24, 1737-1744 (2003).
[CrossRef] [PubMed]

M. A. Holden, S. Kumar, E. T. Castellana, A. Beskok, and P. S. Cremer, "Generating fixed concentration arrays in a microfluidic device," Sens. Actuators B 92, 199-207 (2003).
[CrossRef]

2002 (2)

K. G. Heinze, M. Rarbach, M. Jahnz, and P. Schwille, "Two-photon fluorescence coincidence analysis: rapid measurements of enzyme kinetics," Biophys. J. 83, 1671-1681 (2002).
[CrossRef] [PubMed]

B. H. Kunst, A. Schots, and A. J. W. G. Visser, "Detection of flowing fluorescent particles in a microcapillary using fluorescence correlation spectroscopy," Anal. Chem. 74, 5350-5357 (2002).
[CrossRef] [PubMed]

2001 (4)

J. C. Roulet, R. Völkel, H. P. Herzig, E. Verpoorte, N. F. de Rooij, and R. Dändliker, "Fabrication of multilayer systems combining microfluidic and microoptical elements for fluorescence detection," J. Microelectromech. Syst. 10, 482-491 (2001).
[CrossRef]

K. Dörre, J. Stephan, M. Lapczyna, M. Stuke, H. Dunkel, and M. Eigen, "Highly efficient single molecule detection in microstructures," J. Biotechnol. 86, 225-236 (2001).
[CrossRef] [PubMed]

M. B. Wabuyele, S. M. Ford, J. Barrow, and S. A. Soper, "Single molecule detection of double-stranded DNA in poly(methylmethacrylate) and polycarbonate microfluidic devices," Electrophoresis 22, 3939-3948 (2001).
[CrossRef] [PubMed]

A. Ishijima and T. Yanagida, "Single molecule nanobioscience," Trends Biochem. Sci. 26, 438-444 (2001).
[CrossRef] [PubMed]

2000 (1)

Y. Ishii and T. Yanagida, "Single molecule detection in life science," Single Mol. 1, 5-16 (2000).
[CrossRef]

1999 (4)

A. Van Orden, H. Cai, P. M. Goodwin, and R. A. Keller, "Efficient detection of single DNA fragments in flowing sample streams by two photon fluorescence excitation," Anal. Chem. 71, 2108-2116 (1999).
[CrossRef] [PubMed]

T. Winkler, U. Kettling, A. Koltermann, and M. Eigen, "Confocal fluorescence coincidence analysis: an approach to ultra high-throughput screening," Proc. Natl. Acad. Sci. U.S.A. 96, 1375-1378 (1999).
[CrossRef] [PubMed]

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, M. Sauer, S. Siebert, and J. Wolfrum, "Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC)," Rev. Sci. Instrum. 70, 1835-1841 (1999).
[CrossRef]

B. B. Haab and R. A. Mathies, "Single-molecule detection of DNA separations in microfabricated capillary electrophoresis chips employing focused molecular streams," Anal. Chem. 71, 5137-5145 (1999).
[CrossRef]

1998 (5)

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

C. Zander, K. H. Drexhage, K.-T. Han, J. Wolfrum, and M. Sauer, "Single-molecule counting and identification in a microcapillary," Chem. Phys. Lett. 286, 457-465 (1998).
[CrossRef]

A. Koltermann, U. Kettling, J. Bieschke, T. Winkler, and M. Eigen, "Rapid assay processing by integration of dual-color fluorescence cross-correlation spectroscopy: High throughput screening for enzyme activity," Proc. Natl. Acad. Sci. U.S.A. 95, 1421-1426 (1998).
[CrossRef] [PubMed]

J. M. Song, T. Inoue, H. Kawazumi, and T. Ogama, "Single molecule detection by laser two-photon excited fluorescence in a capillary flowing cell," Anal. Sci. 14, 913-916 (1998).
[CrossRef]

L. Tao and R. T. Kennedy, "Laser-induced fluorescence detection in microcolumn separations," Trends Analyt. Chem. 17, 484-491 (1998).
[CrossRef]

1997 (2)

S. Nie and R. N. Zare, "Optical detection of single molecules," Annu. Rev. Biophys. Biomol. Struct. 26, 567-596 (1997).
[CrossRef] [PubMed]

H. Noji, R. Yasuda, M. Yoshida, and K. Kinosita, Jr., "Direct observation of the rotation of F1-ATPase," Nature 386, 299-302 (1997).
[CrossRef] [PubMed]

1995 (2)

T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, and T. Yanagida, "Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution," Nature 374, 555-559 (1995).
[CrossRef] [PubMed]

L. Q. Li and L. M. Davis, "Rapid and efficient detection of single chromophore molecules in aqueous solution," Appl. Opt. 34, 3208-3217 (1995).
[CrossRef] [PubMed]

1994 (1)

U. Mets and R. Rigler, "Submillisecond detection of single rhodamine molecules in water," J. Fluoresc. 4, 259-264 (1994).
[CrossRef]

1991 (1)

1990 (1)

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

1987 (1)

1984 (1)

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

1976 (2)

S. R. Aragón and R. Pecora, "Fluorescence correlation spectroscopy as a probe of molecular dynamics," J. Chem. Phys. 64, 1791-1803 (1976).
[CrossRef]

T. Hirschfeld, "Optical microscopic observation of single small molecules," Appl. Opt. 15, 2965-2966 (1976).
[CrossRef] [PubMed]

1974 (1)

E. L. Elson and D. Magde, "Fluorescence correlation spectroscopy. I. Conceptual basis and theory," Biopolymers 13, 1-27 (1974).
[CrossRef]

1972 (1)

D. Magde, E. L. Elson, and W. W. Webb, "Thermodynamic fluctuations in a reacting system--measurement by fluorescence correlation spectroscopy," Phys. Rev. Lett. 29, 705-708 (1972).
[CrossRef]

Aragón, S. R.

S. R. Aragón and R. Pecora, "Fluorescence correlation spectroscopy as a probe of molecular dynamics," J. Chem. Phys. 64, 1791-1803 (1976).
[CrossRef]

Ball, D. A.

L. M. Davis, G. Q. Shen, and D. A. Ball, "Saturation effects in fluorescence correlation spectroscopy," in Multiphoton Microscopy in the Biomedical Sciences, A. Periasamy, P. T. C. So, eds., Proc. SPIE 5700, 128-137 (2005).
[CrossRef]

L. M. Davis, P. E. Williams, D. A. Ball, E. D. Matayoshi, and K. M. Swift, "Data reduction methods for application of fluorescence correlation spectroscopy to pharmaceutical drug discovery," Curr. Pharm. Biotechnol. 4, 451-462 (2003).
[CrossRef] [PubMed]

L. M. Davis, G. Shen, and D. A. Ball, "Scanning fluorescence fluctuation spectroscopy for molecular brightness assays," presented at the 49th Annual Meeting of the Biophysical Society, Long Beach, Calif., 12-15 Feb. 2005.

Barrett, L. M.

A. J. Skulan, L. M. Barrett, A. K. Singh, E. B. Cummings, and G. J. Flechtner, "Fabrication and analysis of spatially uniform field electrokinetic flow devices: theory and experiment," Anal. Chem. 77, 6790-6797 (2005).
[CrossRef] [PubMed]

Barrow, J.

M. B. Wabuyele, S. M. Ford, J. Barrow, and S. A. Soper, "Single molecule detection of double-stranded DNA in poly(methylmethacrylate) and polycarbonate microfluidic devices," Electrophoresis 22, 3939-3948 (2001).
[CrossRef] [PubMed]

Becker, W.

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, M. Sauer, S. Siebert, and J. Wolfrum, "Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC)," Rev. Sci. Instrum. 70, 1835-1841 (1999).
[CrossRef]

Beskok, A.

M. A. Holden, S. Kumar, E. T. Castellana, A. Beskok, and P. S. Cremer, "Generating fixed concentration arrays in a microfluidic device," Sens. Actuators B 92, 199-207 (2003).
[CrossRef]

Bieschke, J.

A. Koltermann, U. Kettling, J. Bieschke, T. Winkler, and M. Eigen, "Rapid assay processing by integration of dual-color fluorescence cross-correlation spectroscopy: High throughput screening for enzyme activity," Proc. Natl. Acad. Sci. U.S.A. 95, 1421-1426 (1998).
[CrossRef] [PubMed]

Bischoff, R.

L. A. A. de Jong, D. R. A. Uges, J. P. Franke, and R. Bischoff, "Receptor-ligand binding assays: technologies and applications," J. Chromatogr. B: Biomed Sci. Appl. 829, 1-25 (2005).
[CrossRef]

Cai, H.

A. Van Orden, H. Cai, P. M. Goodwin, and R. A. Keller, "Efficient detection of single DNA fragments in flowing sample streams by two photon fluorescence excitation," Anal. Chem. 71, 2108-2116 (1999).
[CrossRef] [PubMed]

Castellana, E. T.

M. A. Holden, S. Kumar, E. T. Castellana, A. Beskok, and P. S. Cremer, "Generating fixed concentration arrays in a microfluidic device," Sens. Actuators B 92, 199-207 (2003).
[CrossRef]

Chiu, D. T.

J. P. Shelby and D. T. Chiu, "Mapping fast flows over micrometer-length scales using flow-tagging velocimetry and single-molecule detection," Anal. Chem. 75, 1387-1392 (2003).
[CrossRef] [PubMed]

A. Lundqvist, D. T. Chiu, and O. Orwar, "Electrophoretic separation and confocal laser-induced fluorescence detection at ultralow concentrations in constricted fused-silica capillaries," Electrophoresis 24, 1737-1744 (2003).
[CrossRef] [PubMed]

Craighead, H. G.

S. M. Stavis, J. B. Edel, K. T. Samiee, and H. G. Craighead, "Single molecule studies of quantum dot conjugates in a submicrometer fluidic channel," Lab Chip 5, 337-343 (2005).
[CrossRef] [PubMed]

Cremer, P. S.

M. A. Holden, S. Kumar, E. T. Castellana, A. Beskok, and P. S. Cremer, "Generating fixed concentration arrays in a microfluidic device," Sens. Actuators B 92, 199-207 (2003).
[CrossRef]

Cummings, E. B.

A. J. Skulan, L. M. Barrett, A. K. Singh, E. B. Cummings, and G. J. Flechtner, "Fabrication and analysis of spatially uniform field electrokinetic flow devices: theory and experiment," Anal. Chem. 77, 6790-6797 (2005).
[CrossRef] [PubMed]

Dändliker, R.

J. C. Roulet, R. Völkel, H. P. Herzig, E. Verpoorte, N. F. de Rooij, and R. Dändliker, "Fabrication of multilayer systems combining microfluidic and microoptical elements for fluorescence detection," J. Microelectromech. Syst. 10, 482-491 (2001).
[CrossRef]

Davis, L. M.

L. M. Davis, G. Q. Shen, and D. A. Ball, "Saturation effects in fluorescence correlation spectroscopy," in Multiphoton Microscopy in the Biomedical Sciences, A. Periasamy, P. T. C. So, eds., Proc. SPIE 5700, 128-137 (2005).
[CrossRef]

L. M. Davis, P. E. Williams, D. A. Ball, E. D. Matayoshi, and K. M. Swift, "Data reduction methods for application of fluorescence correlation spectroscopy to pharmaceutical drug discovery," Curr. Pharm. Biotechnol. 4, 451-462 (2003).
[CrossRef] [PubMed]

J. C. Fister III, S. C. Jacobson, L. M. Davis, and 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 and L. M. Davis, "Rapid and efficient detection of single chromophore molecules in aqueous solution," Appl. Opt. 34, 3208-3217 (1995).
[CrossRef] [PubMed]

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

L. M. Davis, G. Shen, and D. A. Ball, "Scanning fluorescence fluctuation spectroscopy for molecular brightness assays," presented at the 49th Annual Meeting of the Biophysical Society, Long Beach, Calif., 12-15 Feb. 2005.

de Jong, L. A. A.

L. A. A. de Jong, D. R. A. Uges, J. P. Franke, and R. Bischoff, "Receptor-ligand binding assays: technologies and applications," J. Chromatogr. B: Biomed Sci. Appl. 829, 1-25 (2005).
[CrossRef]

de Rooij, N. F.

J. C. Roulet, R. Völkel, H. P. Herzig, E. Verpoorte, N. F. de Rooij, and R. Dändliker, "Fabrication of multilayer systems combining microfluidic and microoptical elements for fluorescence detection," J. Microelectromech. Syst. 10, 482-491 (2001).
[CrossRef]

Dittrich, P. S.

P. S. Dittrich and A. Manz, "Single-molecule fluorescence detection in microfluidic channels--the Holy Grail in μTAS," Anal. Bioanal. Chem. 382, 1771-1782 (2005).
[CrossRef] [PubMed]

P. S. Dittrich and P. Schwille, "An integrated microfluidic system for reaction, high-sensitivity detection, and sorting of fluorescent cells and particles," Anal. Chem. 75, 5767-5774 (2003).
[CrossRef] [PubMed]

Dörre, K.

K. Dörre, J. Stephan, M. Lapczyna, M. Stuke, H. Dunkel, and M. Eigen, "Highly efficient single molecule detection in microstructures," J. Biotechnol. 86, 225-236 (2001).
[CrossRef] [PubMed]

Dovichi, N. J.

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

Drexhage, K. H.

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, M. Sauer, S. Siebert, and J. Wolfrum, "Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC)," Rev. Sci. Instrum. 70, 1835-1841 (1999).
[CrossRef]

C. Zander, K. H. Drexhage, K.-T. Han, J. Wolfrum, and M. Sauer, "Single-molecule counting and identification in a microcapillary," Chem. Phys. Lett. 286, 457-465 (1998).
[CrossRef]

Dunkel, H.

K. Dörre, J. Stephan, M. Lapczyna, M. Stuke, H. Dunkel, and M. Eigen, "Highly efficient single molecule detection in microstructures," J. Biotechnol. 86, 225-236 (2001).
[CrossRef] [PubMed]

Edel, J. B.

S. M. Stavis, J. B. Edel, K. T. Samiee, and H. G. Craighead, "Single molecule studies of quantum dot conjugates in a submicrometer fluidic channel," Lab Chip 5, 337-343 (2005).
[CrossRef] [PubMed]

Eigen, M.

K. Dörre, J. Stephan, M. Lapczyna, M. Stuke, H. Dunkel, and M. Eigen, "Highly efficient single molecule detection in microstructures," J. Biotechnol. 86, 225-236 (2001).
[CrossRef] [PubMed]

T. Winkler, U. Kettling, A. Koltermann, and M. Eigen, "Confocal fluorescence coincidence analysis: an approach to ultra high-throughput screening," Proc. Natl. Acad. Sci. U.S.A. 96, 1375-1378 (1999).
[CrossRef] [PubMed]

A. Koltermann, U. Kettling, J. Bieschke, T. Winkler, and M. Eigen, "Rapid assay processing by integration of dual-color fluorescence cross-correlation spectroscopy: High throughput screening for enzyme activity," Proc. Natl. Acad. Sci. U.S.A. 95, 1421-1426 (1998).
[CrossRef] [PubMed]

Elson, E. L.

H. Qian and E. L. Elson, "Analysis of confocal laser optics for 3D fluorescence correlation spectroscopy," Appl. Opt. 30, 1185-1195 (1991).
[CrossRef] [PubMed]

E. L. Elson and D. Magde, "Fluorescence correlation spectroscopy. I. Conceptual basis and theory," Biopolymers 13, 1-27 (1974).
[CrossRef]

D. Magde, E. L. Elson, and W. W. Webb, "Thermodynamic fluctuations in a reacting system--measurement by fluorescence correlation spectroscopy," Phys. Rev. Lett. 29, 705-708 (1972).
[CrossRef]

Fister, J. C.

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

Flechtner, G. J.

A. J. Skulan, L. M. Barrett, A. K. Singh, E. B. Cummings, and G. J. Flechtner, "Fabrication and analysis of spatially uniform field electrokinetic flow devices: theory and experiment," Anal. Chem. 77, 6790-6797 (2005).
[CrossRef] [PubMed]

Ford, S. M.

M. B. Wabuyele, S. M. Ford, J. Barrow, and S. A. Soper, "Single molecule detection of double-stranded DNA in poly(methylmethacrylate) and polycarbonate microfluidic devices," Electrophoresis 22, 3939-3948 (2001).
[CrossRef] [PubMed]

Franke, J. P.

L. A. A. de Jong, D. R. A. Uges, J. P. Franke, and R. Bischoff, "Receptor-ligand binding assays: technologies and applications," J. Chromatogr. B: Biomed Sci. Appl. 829, 1-25 (2005).
[CrossRef]

Funatsu, T.

T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, and T. Yanagida, "Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution," Nature 374, 555-559 (1995).
[CrossRef] [PubMed]

Goodwin, P. M.

A. Van Orden, H. Cai, P. M. Goodwin, and R. A. Keller, "Efficient detection of single DNA fragments in flowing sample streams by two photon fluorescence excitation," Anal. Chem. 71, 2108-2116 (1999).
[CrossRef] [PubMed]

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K. O. Greulich, "Single molecule techniques for biomedicine and pharmacology," Curr. Pharm. Biotechnol. 5, 243-259 (2004).
[CrossRef] [PubMed]

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B. B. Haab and R. A. Mathies, "Single-molecule detection of DNA separations in microfabricated capillary electrophoresis chips employing focused molecular streams," Anal. Chem. 71, 5137-5145 (1999).
[CrossRef]

Han, K.-T.

C. Zander, K. H. Drexhage, K.-T. Han, J. Wolfrum, and M. Sauer, "Single-molecule counting and identification in a microcapillary," Chem. Phys. Lett. 286, 457-465 (1998).
[CrossRef]

Harada, Y.

T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, and T. Yanagida, "Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution," Nature 374, 555-559 (1995).
[CrossRef] [PubMed]

Heinze, K. G.

K. G. Heinze, M. Rarbach, M. Jahnz, and P. Schwille, "Two-photon fluorescence coincidence analysis: rapid measurements of enzyme kinetics," Biophys. J. 83, 1671-1681 (2002).
[CrossRef] [PubMed]

Herzig, H. P.

J. C. Roulet, R. Völkel, H. P. Herzig, E. Verpoorte, N. F. de Rooij, and R. Dändliker, "Fabrication of multilayer systems combining microfluidic and microoptical elements for fluorescence detection," J. Microelectromech. Syst. 10, 482-491 (2001).
[CrossRef]

Hickl, H.

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, M. Sauer, S. Siebert, and J. Wolfrum, "Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC)," Rev. Sci. Instrum. 70, 1835-1841 (1999).
[CrossRef]

Hirschfeld, T.

Holden, M. A.

M. A. Holden, S. Kumar, E. T. Castellana, A. Beskok, and P. S. Cremer, "Generating fixed concentration arrays in a microfluidic device," Sens. Actuators B 92, 199-207 (2003).
[CrossRef]

Inoue, T.

J. M. Song, T. Inoue, H. Kawazumi, and T. Ogama, "Single molecule detection by laser two-photon excited fluorescence in a capillary flowing cell," Anal. Sci. 14, 913-916 (1998).
[CrossRef]

Ishii, Y.

Y. Ishii and T. Yanagida, "Single molecule detection in life science," Single Mol. 1, 5-16 (2000).
[CrossRef]

Ishijima, A.

A. Ishijima and T. Yanagida, "Single molecule nanobioscience," Trends Biochem. Sci. 26, 438-444 (2001).
[CrossRef] [PubMed]

Jacobson, S. C.

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

Jahnz, M.

K. G. Heinze, M. Rarbach, M. Jahnz, and P. Schwille, "Two-photon fluorescence coincidence analysis: rapid measurements of enzyme kinetics," Biophys. J. 83, 1671-1681 (2002).
[CrossRef] [PubMed]

Jarvius, J.

J. Melin, H. Johansson, O. Soderberg, F. Nikolajeff, U. Landegren, M. Nilsson, and J. Jarvius, "Thermoplastic microfluidic platform for single-molecule detection, cell culture, and actuation," Anal. Chem. 77, 7122-7130 (2005).
[CrossRef] [PubMed]

Jett, J. H.

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

Johansson, H.

J. Melin, H. Johansson, O. Soderberg, F. Nikolajeff, U. Landegren, M. Nilsson, and J. Jarvius, "Thermoplastic microfluidic platform for single-molecule detection, cell culture, and actuation," Anal. Chem. 77, 7122-7130 (2005).
[CrossRef] [PubMed]

Johnson, M. E.

M. E. Johnson and J. P. Landers, "Fundamentals and practice for ultrasensitive laser-induced fluorescence detection in microanalytical systems," Electrophoresis 25, 3513-3527 (2004).
[CrossRef] [PubMed]

Kawazumi, H.

J. M. Song, T. Inoue, H. Kawazumi, and T. Ogama, "Single molecule detection by laser two-photon excited fluorescence in a capillary flowing cell," Anal. Sci. 14, 913-916 (1998).
[CrossRef]

Keller, R. A.

A. Van Orden, H. Cai, P. M. Goodwin, and R. A. Keller, "Efficient detection of single DNA fragments in flowing sample streams by two photon fluorescence excitation," Anal. Chem. 71, 2108-2116 (1999).
[CrossRef] [PubMed]

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

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

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

Kennedy, R. T.

L. Tao and R. T. Kennedy, "Laser-induced fluorescence detection in microcolumn separations," Trends Analyt. Chem. 17, 484-491 (1998).
[CrossRef]

Kettling, U.

T. Winkler, U. Kettling, A. Koltermann, and M. Eigen, "Confocal fluorescence coincidence analysis: an approach to ultra high-throughput screening," Proc. Natl. Acad. Sci. U.S.A. 96, 1375-1378 (1999).
[CrossRef] [PubMed]

A. Koltermann, U. Kettling, J. Bieschke, T. Winkler, and M. Eigen, "Rapid assay processing by integration of dual-color fluorescence cross-correlation spectroscopy: High throughput screening for enzyme activity," Proc. Natl. Acad. Sci. U.S.A. 95, 1421-1426 (1998).
[CrossRef] [PubMed]

Kinosita, K.

H. Noji, R. Yasuda, M. Yoshida, and K. Kinosita, Jr., "Direct observation of the rotation of F1-ATPase," Nature 386, 299-302 (1997).
[CrossRef] [PubMed]

Koltermann, A.

T. Winkler, U. Kettling, A. Koltermann, and M. Eigen, "Confocal fluorescence coincidence analysis: an approach to ultra high-throughput screening," Proc. Natl. Acad. Sci. U.S.A. 96, 1375-1378 (1999).
[CrossRef] [PubMed]

A. Koltermann, U. Kettling, J. Bieschke, T. Winkler, and M. Eigen, "Rapid assay processing by integration of dual-color fluorescence cross-correlation spectroscopy: High throughput screening for enzyme activity," Proc. Natl. Acad. Sci. U.S.A. 95, 1421-1426 (1998).
[CrossRef] [PubMed]

Kumar, S.

M. A. Holden, S. Kumar, E. T. Castellana, A. Beskok, and P. S. Cremer, "Generating fixed concentration arrays in a microfluidic device," Sens. Actuators B 92, 199-207 (2003).
[CrossRef]

Kunst, B. H.

B. H. Kunst, A. Schots, and A. J. W. G. Visser, "Design of a confocal microfluidic particle sorter using fluorescent photon burst detection," Rev. Sci. Instrum. 75, 2892-2898 (2004).
[CrossRef]

B. H. Kunst, A. Schots, and A. J. W. G. Visser, "Detection of flowing fluorescent particles in a microcapillary using fluorescence correlation spectroscopy," Anal. Chem. 74, 5350-5357 (2002).
[CrossRef] [PubMed]

Landegren, U.

J. Melin, H. Johansson, O. Soderberg, F. Nikolajeff, U. Landegren, M. Nilsson, and J. Jarvius, "Thermoplastic microfluidic platform for single-molecule detection, cell culture, and actuation," Anal. Chem. 77, 7122-7130 (2005).
[CrossRef] [PubMed]

Landers, J. P.

M. E. Johnson and J. P. Landers, "Fundamentals and practice for ultrasensitive laser-induced fluorescence detection in microanalytical systems," Electrophoresis 25, 3513-3527 (2004).
[CrossRef] [PubMed]

Lapczyna, M.

K. Dörre, J. Stephan, M. Lapczyna, M. Stuke, H. Dunkel, and M. Eigen, "Highly efficient single molecule detection in microstructures," J. Biotechnol. 86, 225-236 (2001).
[CrossRef] [PubMed]

Li, L. Q.

Lundqvist, A.

A. Lundqvist, D. T. Chiu, and O. Orwar, "Electrophoretic separation and confocal laser-induced fluorescence detection at ultralow concentrations in constricted fused-silica capillaries," Electrophoresis 24, 1737-1744 (2003).
[CrossRef] [PubMed]

Magde, D.

E. L. Elson and D. Magde, "Fluorescence correlation spectroscopy. I. Conceptual basis and theory," Biopolymers 13, 1-27 (1974).
[CrossRef]

D. Magde, E. L. Elson, and W. W. Webb, "Thermodynamic fluctuations in a reacting system--measurement by fluorescence correlation spectroscopy," Phys. Rev. Lett. 29, 705-708 (1972).
[CrossRef]

Manz, A.

P. S. Dittrich and A. Manz, "Single-molecule fluorescence detection in microfluidic channels--the Holy Grail in μTAS," Anal. Bioanal. Chem. 382, 1771-1782 (2005).
[CrossRef] [PubMed]

Martin, J. C.

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

Matayoshi, E. D.

L. M. Davis, P. E. Williams, D. A. Ball, E. D. Matayoshi, and K. M. Swift, "Data reduction methods for application of fluorescence correlation spectroscopy to pharmaceutical drug discovery," Curr. Pharm. Biotechnol. 4, 451-462 (2003).
[CrossRef] [PubMed]

Mathies, R. A.

B. B. Haab and R. A. Mathies, "Single-molecule detection of DNA separations in microfabricated capillary electrophoresis chips employing focused molecular streams," Anal. Chem. 71, 5137-5145 (1999).
[CrossRef]

Melin, J.

J. Melin, H. Johansson, O. Soderberg, F. Nikolajeff, U. Landegren, M. Nilsson, and J. Jarvius, "Thermoplastic microfluidic platform for single-molecule detection, cell culture, and actuation," Anal. Chem. 77, 7122-7130 (2005).
[CrossRef] [PubMed]

Mets, U.

U. Mets and R. Rigler, "Submillisecond detection of single rhodamine molecules in water," J. Fluoresc. 4, 259-264 (1994).
[CrossRef]

R. Rigler, J. Widengren, and U. Mets, "Interactions and kinetics of single molecules as observed by fluorescence correlation spectroscopy," in Fluorescence Spectroscopy: New Methods and Applications, O.S.Wolfbeis, ed. (Springer-Verlag, 1992), pp. 13-24.

Nie, S.

S. Nie and R. N. Zare, "Optical detection of single molecules," Annu. Rev. Biophys. Biomol. Struct. 26, 567-596 (1997).
[CrossRef] [PubMed]

Nikolajeff, F.

J. Melin, H. Johansson, O. Soderberg, F. Nikolajeff, U. Landegren, M. Nilsson, and J. Jarvius, "Thermoplastic microfluidic platform for single-molecule detection, cell culture, and actuation," Anal. Chem. 77, 7122-7130 (2005).
[CrossRef] [PubMed]

Nilsson, M.

J. Melin, H. Johansson, O. Soderberg, F. Nikolajeff, U. Landegren, M. Nilsson, and J. Jarvius, "Thermoplastic microfluidic platform for single-molecule detection, cell culture, and actuation," Anal. Chem. 77, 7122-7130 (2005).
[CrossRef] [PubMed]

Noji, H.

H. Noji, R. Yasuda, M. Yoshida, and K. Kinosita, Jr., "Direct observation of the rotation of F1-ATPase," Nature 386, 299-302 (1997).
[CrossRef] [PubMed]

Nquyen, D. C.

Ogama, T.

J. M. Song, T. Inoue, H. Kawazumi, and T. Ogama, "Single molecule detection by laser two-photon excited fluorescence in a capillary flowing cell," Anal. Sci. 14, 913-916 (1998).
[CrossRef]

Orwar, O.

A. Lundqvist, D. T. Chiu, and O. Orwar, "Electrophoretic separation and confocal laser-induced fluorescence detection at ultralow concentrations in constricted fused-silica capillaries," Electrophoresis 24, 1737-1744 (2003).
[CrossRef] [PubMed]

Pecora, R.

S. R. Aragón and R. Pecora, "Fluorescence correlation spectroscopy as a probe of molecular dynamics," J. Chem. Phys. 64, 1791-1803 (1976).
[CrossRef]

Qian, H.

Ramsey, J. M.

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

Rarbach, M.

K. G. Heinze, M. Rarbach, M. Jahnz, and P. Schwille, "Two-photon fluorescence coincidence analysis: rapid measurements of enzyme kinetics," Biophys. J. 83, 1671-1681 (2002).
[CrossRef] [PubMed]

Rigler, R.

U. Mets and R. Rigler, "Submillisecond detection of single rhodamine molecules in water," J. Fluoresc. 4, 259-264 (1994).
[CrossRef]

R. Rigler, J. Widengren, and U. Mets, "Interactions and kinetics of single molecules as observed by fluorescence correlation spectroscopy," in Fluorescence Spectroscopy: New Methods and Applications, O.S.Wolfbeis, ed. (Springer-Verlag, 1992), pp. 13-24.

Roulet, J. C.

J. C. Roulet, R. Völkel, H. P. Herzig, E. Verpoorte, N. F. de Rooij, and R. Dändliker, "Fabrication of multilayer systems combining microfluidic and microoptical elements for fluorescence detection," J. Microelectromech. Syst. 10, 482-491 (2001).
[CrossRef]

Saito, K.

T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, and T. Yanagida, "Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution," Nature 374, 555-559 (1995).
[CrossRef] [PubMed]

Samiee, K. T.

S. M. Stavis, J. B. Edel, K. T. Samiee, and H. G. Craighead, "Single molecule studies of quantum dot conjugates in a submicrometer fluidic channel," Lab Chip 5, 337-343 (2005).
[CrossRef] [PubMed]

Sauer, M.

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, M. Sauer, S. Siebert, and J. Wolfrum, "Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC)," Rev. Sci. Instrum. 70, 1835-1841 (1999).
[CrossRef]

C. Zander, K. H. Drexhage, K.-T. Han, J. Wolfrum, and M. Sauer, "Single-molecule counting and identification in a microcapillary," Chem. Phys. Lett. 286, 457-465 (1998).
[CrossRef]

Schots, A.

B. H. Kunst, A. Schots, and A. J. W. G. Visser, "Design of a confocal microfluidic particle sorter using fluorescent photon burst detection," Rev. Sci. Instrum. 75, 2892-2898 (2004).
[CrossRef]

B. H. Kunst, A. Schots, and A. J. W. G. Visser, "Detection of flowing fluorescent particles in a microcapillary using fluorescence correlation spectroscopy," Anal. Chem. 74, 5350-5357 (2002).
[CrossRef] [PubMed]

Schwille, P.

P. S. Dittrich and P. Schwille, "An integrated microfluidic system for reaction, high-sensitivity detection, and sorting of fluorescent cells and particles," Anal. Chem. 75, 5767-5774 (2003).
[CrossRef] [PubMed]

K. G. Heinze, M. Rarbach, M. Jahnz, and P. Schwille, "Two-photon fluorescence coincidence analysis: rapid measurements of enzyme kinetics," Biophys. J. 83, 1671-1681 (2002).
[CrossRef] [PubMed]

Seitzinger, N. K.

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

Shelby, J. P.

J. P. Shelby and D. T. Chiu, "Mapping fast flows over micrometer-length scales using flow-tagging velocimetry and single-molecule detection," Anal. Chem. 75, 1387-1392 (2003).
[CrossRef] [PubMed]

Shen, G.

L. M. Davis, G. Shen, and D. A. Ball, "Scanning fluorescence fluctuation spectroscopy for molecular brightness assays," presented at the 49th Annual Meeting of the Biophysical Society, Long Beach, Calif., 12-15 Feb. 2005.

Shen, G. Q.

L. M. Davis, G. Q. Shen, and D. A. Ball, "Saturation effects in fluorescence correlation spectroscopy," in Multiphoton Microscopy in the Biomedical Sciences, A. Periasamy, P. T. C. So, eds., Proc. SPIE 5700, 128-137 (2005).
[CrossRef]

Shera, E. B.

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

Siebert, S.

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, M. Sauer, S. Siebert, and J. Wolfrum, "Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC)," Rev. Sci. Instrum. 70, 1835-1841 (1999).
[CrossRef]

Singh, A. K.

A. J. Skulan, L. M. Barrett, A. K. Singh, E. B. Cummings, and G. J. Flechtner, "Fabrication and analysis of spatially uniform field electrokinetic flow devices: theory and experiment," Anal. Chem. 77, 6790-6797 (2005).
[CrossRef] [PubMed]

Skulan, A. J.

A. J. Skulan, L. M. Barrett, A. K. Singh, E. B. Cummings, and G. J. Flechtner, "Fabrication and analysis of spatially uniform field electrokinetic flow devices: theory and experiment," Anal. Chem. 77, 6790-6797 (2005).
[CrossRef] [PubMed]

Soderberg, O.

J. Melin, H. Johansson, O. Soderberg, F. Nikolajeff, U. Landegren, M. Nilsson, and J. Jarvius, "Thermoplastic microfluidic platform for single-molecule detection, cell culture, and actuation," Anal. Chem. 77, 7122-7130 (2005).
[CrossRef] [PubMed]

Song, J. M.

J. M. Song, T. Inoue, H. Kawazumi, and T. Ogama, "Single molecule detection by laser two-photon excited fluorescence in a capillary flowing cell," Anal. Sci. 14, 913-916 (1998).
[CrossRef]

Soper, S. A.

M. B. Wabuyele, S. M. Ford, J. Barrow, and S. A. Soper, "Single molecule detection of double-stranded DNA in poly(methylmethacrylate) and polycarbonate microfluidic devices," Electrophoresis 22, 3939-3948 (2001).
[CrossRef] [PubMed]

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

Stavis, S. M.

S. M. Stavis, J. B. Edel, K. T. Samiee, and H. G. Craighead, "Single molecule studies of quantum dot conjugates in a submicrometer fluidic channel," Lab Chip 5, 337-343 (2005).
[CrossRef] [PubMed]

Stephan, J.

K. Dörre, J. Stephan, M. Lapczyna, M. Stuke, H. Dunkel, and M. Eigen, "Highly efficient single molecule detection in microstructures," J. Biotechnol. 86, 225-236 (2001).
[CrossRef] [PubMed]

Stuke, M.

K. Dörre, J. Stephan, M. Lapczyna, M. Stuke, H. Dunkel, and M. Eigen, "Highly efficient single molecule detection in microstructures," J. Biotechnol. 86, 225-236 (2001).
[CrossRef] [PubMed]

Swift, K. M.

L. M. Davis, P. E. Williams, D. A. Ball, E. D. Matayoshi, and K. M. Swift, "Data reduction methods for application of fluorescence correlation spectroscopy to pharmaceutical drug discovery," Curr. Pharm. Biotechnol. 4, 451-462 (2003).
[CrossRef] [PubMed]

Tao, L.

L. Tao and R. T. Kennedy, "Laser-induced fluorescence detection in microcolumn separations," Trends Analyt. Chem. 17, 484-491 (1998).
[CrossRef]

Tokunaga, M.

T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, and T. Yanagida, "Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution," Nature 374, 555-559 (1995).
[CrossRef] [PubMed]

Trkula, M.

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

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

Uges, D. R. A.

L. A. A. de Jong, D. R. A. Uges, J. P. Franke, and R. Bischoff, "Receptor-ligand binding assays: technologies and applications," J. Chromatogr. B: Biomed Sci. Appl. 829, 1-25 (2005).
[CrossRef]

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A. Van Orden, H. Cai, P. M. Goodwin, and R. A. Keller, "Efficient detection of single DNA fragments in flowing sample streams by two photon fluorescence excitation," Anal. Chem. 71, 2108-2116 (1999).
[CrossRef] [PubMed]

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J. C. Roulet, R. Völkel, H. P. Herzig, E. Verpoorte, N. F. de Rooij, and R. Dändliker, "Fabrication of multilayer systems combining microfluidic and microoptical elements for fluorescence detection," J. Microelectromech. Syst. 10, 482-491 (2001).
[CrossRef]

Visser, A. J. W. G.

B. H. Kunst, A. Schots, and A. J. W. G. Visser, "Design of a confocal microfluidic particle sorter using fluorescent photon burst detection," Rev. Sci. Instrum. 75, 2892-2898 (2004).
[CrossRef]

B. H. Kunst, A. Schots, and A. J. W. G. Visser, "Detection of flowing fluorescent particles in a microcapillary using fluorescence correlation spectroscopy," Anal. Chem. 74, 5350-5357 (2002).
[CrossRef] [PubMed]

Völkel, R.

J. C. Roulet, R. Völkel, H. P. Herzig, E. Verpoorte, N. F. de Rooij, and R. Dändliker, "Fabrication of multilayer systems combining microfluidic and microoptical elements for fluorescence detection," J. Microelectromech. Syst. 10, 482-491 (2001).
[CrossRef]

Wabuyele, M. B.

M. B. Wabuyele, S. M. Ford, J. Barrow, and S. A. Soper, "Single molecule detection of double-stranded DNA in poly(methylmethacrylate) and polycarbonate microfluidic devices," Electrophoresis 22, 3939-3948 (2001).
[CrossRef] [PubMed]

Webb, W. W.

D. Magde, E. L. Elson, and W. W. Webb, "Thermodynamic fluctuations in a reacting system--measurement by fluorescence correlation spectroscopy," Phys. Rev. Lett. 29, 705-708 (1972).
[CrossRef]

Widengren, J.

R. Rigler, J. Widengren, and U. Mets, "Interactions and kinetics of single molecules as observed by fluorescence correlation spectroscopy," in Fluorescence Spectroscopy: New Methods and Applications, O.S.Wolfbeis, ed. (Springer-Verlag, 1992), pp. 13-24.

Williams, P. E.

L. M. Davis, P. E. Williams, D. A. Ball, E. D. Matayoshi, and K. M. Swift, "Data reduction methods for application of fluorescence correlation spectroscopy to pharmaceutical drug discovery," Curr. Pharm. Biotechnol. 4, 451-462 (2003).
[CrossRef] [PubMed]

Winkler, T.

T. Winkler, U. Kettling, A. Koltermann, and M. Eigen, "Confocal fluorescence coincidence analysis: an approach to ultra high-throughput screening," Proc. Natl. Acad. Sci. U.S.A. 96, 1375-1378 (1999).
[CrossRef] [PubMed]

A. Koltermann, U. Kettling, J. Bieschke, T. Winkler, and M. Eigen, "Rapid assay processing by integration of dual-color fluorescence cross-correlation spectroscopy: High throughput screening for enzyme activity," Proc. Natl. Acad. Sci. U.S.A. 95, 1421-1426 (1998).
[CrossRef] [PubMed]

Wolfrum, J.

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, M. Sauer, S. Siebert, and J. Wolfrum, "Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC)," Rev. Sci. Instrum. 70, 1835-1841 (1999).
[CrossRef]

C. Zander, K. H. Drexhage, K.-T. Han, J. Wolfrum, and M. Sauer, "Single-molecule counting and identification in a microcapillary," Chem. Phys. Lett. 286, 457-465 (1998).
[CrossRef]

Yanagida, T.

A. Ishijima and T. Yanagida, "Single molecule nanobioscience," Trends Biochem. Sci. 26, 438-444 (2001).
[CrossRef] [PubMed]

Y. Ishii and T. Yanagida, "Single molecule detection in life science," Single Mol. 1, 5-16 (2000).
[CrossRef]

T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, and T. Yanagida, "Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution," Nature 374, 555-559 (1995).
[CrossRef] [PubMed]

Yasuda, R.

H. Noji, R. Yasuda, M. Yoshida, and K. Kinosita, Jr., "Direct observation of the rotation of F1-ATPase," Nature 386, 299-302 (1997).
[CrossRef] [PubMed]

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J. J. Zheng and E. S. Yeung, "Counting single DNA molecules in a capillary with radial focusing," Aust. J. Chem. 56, 149-153 (2003).
[CrossRef]

Yoshida, M.

H. Noji, R. Yasuda, M. Yoshida, and K. Kinosita, Jr., "Direct observation of the rotation of F1-ATPase," Nature 386, 299-302 (1997).
[CrossRef] [PubMed]

Zander, C.

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, M. Sauer, S. Siebert, and J. Wolfrum, "Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC)," Rev. Sci. Instrum. 70, 1835-1841 (1999).
[CrossRef]

C. Zander, K. H. Drexhage, K.-T. Han, J. Wolfrum, and M. Sauer, "Single-molecule counting and identification in a microcapillary," Chem. Phys. Lett. 286, 457-465 (1998).
[CrossRef]

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S. Nie and R. N. Zare, "Optical detection of single molecules," Annu. Rev. Biophys. Biomol. Struct. 26, 567-596 (1997).
[CrossRef] [PubMed]

Zheng, J. J.

J. J. Zheng and E. S. Yeung, "Counting single DNA molecules in a capillary with radial focusing," Aust. J. Chem. 56, 149-153 (2003).
[CrossRef]

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P. S. Dittrich and A. Manz, "Single-molecule fluorescence detection in microfluidic channels--the Holy Grail in μTAS," Anal. Bioanal. Chem. 382, 1771-1782 (2005).
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J. Melin, H. Johansson, O. Soderberg, F. Nikolajeff, U. Landegren, M. Nilsson, and J. Jarvius, "Thermoplastic microfluidic platform for single-molecule detection, cell culture, and actuation," Anal. Chem. 77, 7122-7130 (2005).
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B. H. Kunst, A. Schots, and A. J. W. G. Visser, "Detection of flowing fluorescent particles in a microcapillary using fluorescence correlation spectroscopy," Anal. Chem. 74, 5350-5357 (2002).
[CrossRef] [PubMed]

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J. M. Song, T. Inoue, H. Kawazumi, and T. Ogama, "Single molecule detection by laser two-photon excited fluorescence in a capillary flowing cell," Anal. Sci. 14, 913-916 (1998).
[CrossRef]

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S. Nie and R. N. Zare, "Optical detection of single molecules," Annu. Rev. Biophys. Biomol. Struct. 26, 567-596 (1997).
[CrossRef] [PubMed]

Appl. Opt. (3)

Aust. J. Chem. (1)

J. J. Zheng and E. S. Yeung, "Counting single DNA molecules in a capillary with radial focusing," Aust. J. Chem. 56, 149-153 (2003).
[CrossRef]

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K. G. Heinze, M. Rarbach, M. Jahnz, and P. Schwille, "Two-photon fluorescence coincidence analysis: rapid measurements of enzyme kinetics," Biophys. J. 83, 1671-1681 (2002).
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E. L. Elson and D. Magde, "Fluorescence correlation spectroscopy. I. Conceptual basis and theory," Biopolymers 13, 1-27 (1974).
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Chem. Phys. Lett. (2)

C. Zander, K. H. Drexhage, K.-T. Han, J. Wolfrum, and M. Sauer, "Single-molecule counting and identification in a microcapillary," Chem. Phys. Lett. 286, 457-465 (1998).
[CrossRef]

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

Curr. Pharm. Biotechnol. (2)

L. M. Davis, P. E. Williams, D. A. Ball, E. D. Matayoshi, and K. M. Swift, "Data reduction methods for application of fluorescence correlation spectroscopy to pharmaceutical drug discovery," Curr. Pharm. Biotechnol. 4, 451-462 (2003).
[CrossRef] [PubMed]

K. O. Greulich, "Single molecule techniques for biomedicine and pharmacology," Curr. Pharm. Biotechnol. 5, 243-259 (2004).
[CrossRef] [PubMed]

Electrophoresis (3)

M. B. Wabuyele, S. M. Ford, J. Barrow, and S. A. Soper, "Single molecule detection of double-stranded DNA in poly(methylmethacrylate) and polycarbonate microfluidic devices," Electrophoresis 22, 3939-3948 (2001).
[CrossRef] [PubMed]

A. Lundqvist, D. T. Chiu, and O. Orwar, "Electrophoretic separation and confocal laser-induced fluorescence detection at ultralow concentrations in constricted fused-silica capillaries," Electrophoresis 24, 1737-1744 (2003).
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K. Dörre, J. Stephan, M. Lapczyna, M. Stuke, H. Dunkel, and M. Eigen, "Highly efficient single molecule detection in microstructures," J. Biotechnol. 86, 225-236 (2001).
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S. R. Aragón and R. Pecora, "Fluorescence correlation spectroscopy as a probe of molecular dynamics," J. Chem. Phys. 64, 1791-1803 (1976).
[CrossRef]

J. Chromatogr. (1)

L. A. A. de Jong, D. R. A. Uges, J. P. Franke, and R. Bischoff, "Receptor-ligand binding assays: technologies and applications," J. Chromatogr. B: Biomed Sci. Appl. 829, 1-25 (2005).
[CrossRef]

J. Fluoresc. (1)

U. Mets and R. Rigler, "Submillisecond detection of single rhodamine molecules in water," J. Fluoresc. 4, 259-264 (1994).
[CrossRef]

J. Microelectromech. Syst. (1)

J. C. Roulet, R. Völkel, H. P. Herzig, E. Verpoorte, N. F. de Rooij, and R. Dändliker, "Fabrication of multilayer systems combining microfluidic and microoptical elements for fluorescence detection," J. Microelectromech. Syst. 10, 482-491 (2001).
[CrossRef]

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

Lab Chip (1)

S. M. Stavis, J. B. Edel, K. T. Samiee, and H. G. Craighead, "Single molecule studies of quantum dot conjugates in a submicrometer fluidic channel," Lab Chip 5, 337-343 (2005).
[CrossRef] [PubMed]

Nature (2)

T. Funatsu, Y. Harada, M. Tokunaga, K. Saito, and T. Yanagida, "Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution," Nature 374, 555-559 (1995).
[CrossRef] [PubMed]

H. Noji, R. Yasuda, M. Yoshida, and K. Kinosita, Jr., "Direct observation of the rotation of F1-ATPase," Nature 386, 299-302 (1997).
[CrossRef] [PubMed]

Phys. Rev. Lett. (1)

D. Magde, E. L. Elson, and W. W. Webb, "Thermodynamic fluctuations in a reacting system--measurement by fluorescence correlation spectroscopy," Phys. Rev. Lett. 29, 705-708 (1972).
[CrossRef]

Proc. Natl. Acad. Sci. U.S.A. (2)

A. Koltermann, U. Kettling, J. Bieschke, T. Winkler, and M. Eigen, "Rapid assay processing by integration of dual-color fluorescence cross-correlation spectroscopy: High throughput screening for enzyme activity," Proc. Natl. Acad. Sci. U.S.A. 95, 1421-1426 (1998).
[CrossRef] [PubMed]

T. Winkler, U. Kettling, A. Koltermann, and M. Eigen, "Confocal fluorescence coincidence analysis: an approach to ultra high-throughput screening," Proc. Natl. Acad. Sci. U.S.A. 96, 1375-1378 (1999).
[CrossRef] [PubMed]

Proc. SPIE (1)

L. M. Davis, G. Q. Shen, and D. A. Ball, "Saturation effects in fluorescence correlation spectroscopy," in Multiphoton Microscopy in the Biomedical Sciences, A. Periasamy, P. T. C. So, eds., Proc. SPIE 5700, 128-137 (2005).
[CrossRef]

Rev. Sci. Instrum. (2)

W. Becker, H. Hickl, C. Zander, K. H. Drexhage, M. Sauer, S. Siebert, and J. Wolfrum, "Time-resolved detection and identification of single analyte molecules in microcapillaries by time-correlated single-photon counting (TCSPC)," Rev. Sci. Instrum. 70, 1835-1841 (1999).
[CrossRef]

B. H. Kunst, A. Schots, and A. J. W. G. Visser, "Design of a confocal microfluidic particle sorter using fluorescent photon burst detection," Rev. Sci. Instrum. 75, 2892-2898 (2004).
[CrossRef]

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M. A. Holden, S. Kumar, E. T. Castellana, A. Beskok, and P. S. Cremer, "Generating fixed concentration arrays in a microfluidic device," Sens. Actuators B 92, 199-207 (2003).
[CrossRef]

Single Mol. (1)

Y. Ishii and T. Yanagida, "Single molecule detection in life science," Single Mol. 1, 5-16 (2000).
[CrossRef]

Trends Analyt. Chem. (1)

L. Tao and R. T. Kennedy, "Laser-induced fluorescence detection in microcolumn separations," Trends Analyt. Chem. 17, 484-491 (1998).
[CrossRef]

Trends Biochem. Sci. (1)

A. Ishijima and T. Yanagida, "Single molecule nanobioscience," Trends Biochem. Sci. 26, 438-444 (2001).
[CrossRef] [PubMed]

Other (2)

R. Rigler, J. Widengren, and U. Mets, "Interactions and kinetics of single molecules as observed by fluorescence correlation spectroscopy," in Fluorescence Spectroscopy: New Methods and Applications, O.S.Wolfbeis, ed. (Springer-Verlag, 1992), pp. 13-24.

L. M. Davis, G. Shen, and D. A. Ball, "Scanning fluorescence fluctuation spectroscopy for molecular brightness assays," presented at the 49th Annual Meeting of the Biophysical Society, Long Beach, Calif., 12-15 Feb. 2005.

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Figures (7)

Fig. 1
Fig. 1

(Color online) Schematic of the confocal fluorescence microscope and capillary to induce axial flow.

Fig. 2
Fig. 2

CCD image of a 10 μ m capillary during the alignmentprocedure.

Fig. 3
Fig. 3

Background count rate at various distances from the capillary: (a) 5 μ m capillary with a 150 μ m pinhole, laser power of 30 μ W , λ = 585   nm and (b) 10 μ m capillary with a 75 μ m pinhole, laser power of 30 μ W , λ = 585   nm .

Fig. 4
Fig. 4

(Color online) Time-resolved background counts at various distances from the capillary: (a) 5 μ m capillary with a 150 μ m pinhole, laser power of 30 μ W , λ = 585   nm and (b) 10 μ m capillary with a 75 μ m pinhole, laser power of 30 μ W , λ = 585   nm .

Fig. 5
Fig. 5

(Color online) (a) Normalized autocorrelation function (ACF) for 50 pM Alexa Fluor 594 at various distances from the 5 μ m capillary with a 150 μ m pinhole, laser power of 30 μ W , λ = 585   nm . (b) Calculated flow velocities at these distances using the parameters obtained by a fit of the ACF to Eq. (7) along with a fit (solid line), which indicates a quadratic decrease in flow velocity with distance.

Fig. 6
Fig. 6

Plot of count rate versus time for 50 pM Alexa Fluor 594 obtained with the capillary 50 μ m from the laser focus, 5 μm capillary with a 150 μ m pinhole, laser power of 30 μ W , λ = 585   nm .

Fig. 7
Fig. 7

Probability density for the amplitudes of peaks obtained from a Gaussian-weighted sliding sum of the photon data streams with flow and the capillary 50 μ m from the laser focus. The width for the Gaussian weights is σ = τ F , with τ F obtained from a curve fit to the autocorrelation function. Comparison of results for a 50 pM solution of Alexa Fluor 594 and for a 50 pM solution of Alexa Fluor 610 demonstrates the possibility to distinguish species with different brightness. Obtained with a 5 μ m capillary with a 150 μ m pinhole, laser power of 30 μ W , λ = 585   nm .

Equations (8)

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

G ( τ ) = F ( t ) F ( t + τ ) F ( t ) F ( t + τ ) ,
F ( t ) = lim T 1 2 T T T F ( t ) d t .
G d i f f ( τ ) = 1 + α N ( 1 + τ τ D ) 1 ( 1 + χ τ τ D ) 1 / 2 ,
α = γ ( 1 + B N Φ ) 2 ,
χ = ( ω 0 z 0 ) 2 ,
τ D = ω 0 2 / 4 D ,
G flow ( τ ) = 1 + α N [ 1 + τ τ D ] 1 [ 1 + χ τ τ D ] 1 / 2 × exp { ( τ / τ F ) 2 1 + χ τ / τ D } ,
τ F = z 0 / v F ,

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