Abstract

We report on various excitation geometries used in ultrasensitive flow cytometry that yield a linear relation between the fluorescence intensity measured from individual stained DNA fragments and the lengths of the fragments (in base pairs). This linearity holds for DNA samples that exhibit a wide range of conformations. The variety of DNA conformations leads to a distribution of dipole moment orientations for the dye molecules intercalated into the DNA. It is consequently important to use an excitation geometry such that all dye molecules are detected with similar efficiency. To estimate the conformation and the extent of elongation of the stained fragments in the flow, fluorescence polarization anisotropy and autocorrelation measurements were performed. Significant extension was observed for DNA fragments under the flow conditions frequently used for DNA fragment sizing. Classical calculations of the fluorescence emission collected over a finite solid angle are in agreement with the experimental measurements and have confirmed the relative insensitivity to DNA conformation of an orthogonal excitation geometry. Furthermore, the calculations suggested a modified excitation geometry that has increased our sizing resolution.

© 2000 Optical Society of America

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  1. P. M. Goodwin, M. E. Johnson, J. C. Martin, W. P. Ambrose, B. L. Marrone, J. H. Jett, R. A. Keller, “Rapid sizing of individual fluorescently stained DNA fragments by flow cytometry,” Nucleic Acids Res. 21, 803–806 (1993).
    [CrossRef] [PubMed]
  2. M. E. Johnson, P. M. Goodwin, W. P. Ambrose, J. C. Martin, B. L. Marrone, J. H. Jett, R. A. Keller, “Sizing of DNA fragments by flow cytometry,” in Advances in FluorescenceSensing Technology IV, J. R. Lakowicz, S. A. Soper, R. B. Thompson, eds., Proc. SPIE1895, 69–78 (1993).
  3. J. T. Petty, M. E. Johnson, P. M. Goodwin, J. C. Martin, J. H. Jett, R. A. Keller, “Characterization of DNA size determination of small fragments by flow cytometry,” Anal. Chem. 67, 1755–1761 (1995).
    [CrossRef]
  4. Z. Huang, J. T. Petty, B. O’Quinn, J. L. Longmire, N. C. Brown, J. H. Jett, R. A. Keller, “Large DNA fragment sizing by flow cytometry: application to the characterization of P1 artificial chromosome (PAC) clones,” Nucleic Acids Res. 24, 4202–4209 (1996).
    [CrossRef] [PubMed]
  5. Z. Huang, J. H. Jett, R. A. Keller, “Bacteria genome fingerprinting by flow cytometry,” Cytometry 35, 169–175 (1999).
    [CrossRef] [PubMed]
  6. Y. Kim, J. H. Jett, E. J. Larson, J. R. Penttila, B. L. Marrone, R. A. Keller, “Bacterial fingerprinting by flow cytometry: bacterial species discrimination,” Cytometry 36, 1–9 (1999).
    [CrossRef]
  7. W. P. Ambrose, H. Cai, P. M. Goodwin, J. H. Jett, R. C. Habbersett, E. J. Larson, W. K. Grace, J. H. Werner, R. A. Keller, “Flow cytometric sizing of DNA fragments,” in Topics in Fluorescence: DNA Technology, J. R. Lacowicz, ed. (Plenum, New York, 2000), Vol. 7.
  8. A. Castro, F. R. Fairfield, E. B. Shera, “Fluorescence detection and size measurement of single DNA molecules,” Anal. Chem. 65, 849–852 (1993).
    [CrossRef]
  9. A. Castro, E. B. Shera, “Single molecule electrophoresis,” Anal. Chem. 67, 3181–3186 (1995).
    [CrossRef]
  10. J. M. Schins, A. Agronskaia, B. G. de Grooth, J. Greve, “New technique for high resolution DNA sizing in epi-illumination,” Cytometry 32, 132–136 (1998).
    [CrossRef] [PubMed]
  11. A. Agronskaia, A. Florians, K. O. van der Werf, J. M. Schins, B. G. de Grooth, J. Greve, “Photon-counting device compatible with conventional flow cytometric data acquisition electronics,” Cytometry 32, 255–259 (1998).
    [CrossRef] [PubMed]
  12. A. Agronskaia, J. M. Schins, B. G. de Grooth, J. Greve, “Polarization effects in flow cytometric DNA sizing,” Appl. Opt. 38, 714–719 (1999).
    [CrossRef]
  13. H. P. Chou, C. Spence, A. Scherer, S. Quake, “A microfabricated device for sizing and sorting DNA molecules,” Proc. Natl. Acad. Sci. USA 96, 11–13 (1999).
    [CrossRef] [PubMed]
  14. J. Enderlein, D. L. Robbins, W. P. Ambrose, R. A. Keller, “Molecular shot noise, burst size distribution, and single-molecule detection in fluid flow: effects of multiple occupancy,” J. Phys. Chem. 102, 6089–6094 (1998).
    [CrossRef]
  15. A. Van Orden, H. Cai, P. M. Goodwin, 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]
  16. J. P. Jacobsen, J. B. Pedersen, L. F. Hansen, D. E. Wemmer, “Site selective bis-intercalation of a homodimeric thiazole orange dye in DNA oligonucleotides,” Nucleic Acids Res. 23, 753–760 (1995).
    [CrossRef] [PubMed]
  17. H. P. Spielmann, D. E. Wemmer, J. Jacobsen, “Solution structure of a DNA complex with the fluorescent bis-intercalator TOTO determined by NMR spectroscopy,” Biochemistry 34, 8542–8553 (1995).
    [CrossRef] [PubMed]
  18. T. T. Perkins, D. E. Smith, S. Chu, “Single polymer dynamics in an elongational flow,” Science 276, 2016–2021 (1997).
    [CrossRef] [PubMed]
  19. D. E. Smith, H. P. Babcock, S. Chu, “Single polymer dynamics in steady shear flow,” Science 283, 1724–1727 (1999).
    [CrossRef] [PubMed]
  20. J. D. Jackson, Classical Electrodynamics, (Wiley, New York, 1975), p. 138.
  21. A. Larsson, C. Carlsson, M. Jonsson, B. Albinsson, “Characterization of the binding of the fluorescent dyes YO and YOYO to DNA by polarized light spectroscopy,” J. Am. Chem. Soc. 116, 8459–8465 (1994).
    [CrossRef]
  22. C. Carlsson, A. Larsson, M. Bjorkman, M. Jonsson, B. Al-binsson, “Experimental and simulated fluorescence depolarization due to energy transfer as tools to study DNA-dye interactions,” Biopolymers 41, 481–494 (1997).
    [CrossRef]
  23. D. Axelrod, “Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization,” Biophys. J. 26, 557–574 (1979).
    [CrossRef] [PubMed]
  24. D. Axelrod, “Fluorescence polarization microscopy,” in Methods in Cell Biology (Academic, San Diego, Calif., 1989), Vol. 30, pp. 333–352.
    [CrossRef]
  25. C. C. Tsai, S. C. Jain, H. M. Sobell, “X-ray crystallographic visualization of drug-nucleic acid intercalative binding: structure of an ethidium-dinucleoside monophosphate crystalline complex, ethidium: 5-iodouridylyl(3′-5′) adenosine,” Proc. Natl. Acad. Sci. USA 72, 628–632 (1975).
    [CrossRef]

1999 (6)

Z. Huang, J. H. Jett, R. A. Keller, “Bacteria genome fingerprinting by flow cytometry,” Cytometry 35, 169–175 (1999).
[CrossRef] [PubMed]

Y. Kim, J. H. Jett, E. J. Larson, J. R. Penttila, B. L. Marrone, R. A. Keller, “Bacterial fingerprinting by flow cytometry: bacterial species discrimination,” Cytometry 36, 1–9 (1999).
[CrossRef]

H. P. Chou, C. Spence, A. Scherer, S. Quake, “A microfabricated device for sizing and sorting DNA molecules,” Proc. Natl. Acad. Sci. USA 96, 11–13 (1999).
[CrossRef] [PubMed]

A. Van Orden, H. Cai, P. M. Goodwin, 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]

D. E. Smith, H. P. Babcock, S. Chu, “Single polymer dynamics in steady shear flow,” Science 283, 1724–1727 (1999).
[CrossRef] [PubMed]

A. Agronskaia, J. M. Schins, B. G. de Grooth, J. Greve, “Polarization effects in flow cytometric DNA sizing,” Appl. Opt. 38, 714–719 (1999).
[CrossRef]

1998 (3)

J. Enderlein, D. L. Robbins, W. P. Ambrose, R. A. Keller, “Molecular shot noise, burst size distribution, and single-molecule detection in fluid flow: effects of multiple occupancy,” J. Phys. Chem. 102, 6089–6094 (1998).
[CrossRef]

J. M. Schins, A. Agronskaia, B. G. de Grooth, J. Greve, “New technique for high resolution DNA sizing in epi-illumination,” Cytometry 32, 132–136 (1998).
[CrossRef] [PubMed]

A. Agronskaia, A. Florians, K. O. van der Werf, J. M. Schins, B. G. de Grooth, J. Greve, “Photon-counting device compatible with conventional flow cytometric data acquisition electronics,” Cytometry 32, 255–259 (1998).
[CrossRef] [PubMed]

1997 (2)

C. Carlsson, A. Larsson, M. Bjorkman, M. Jonsson, B. Al-binsson, “Experimental and simulated fluorescence depolarization due to energy transfer as tools to study DNA-dye interactions,” Biopolymers 41, 481–494 (1997).
[CrossRef]

T. T. Perkins, D. E. Smith, S. Chu, “Single polymer dynamics in an elongational flow,” Science 276, 2016–2021 (1997).
[CrossRef] [PubMed]

1996 (1)

Z. Huang, J. T. Petty, B. O’Quinn, J. L. Longmire, N. C. Brown, J. H. Jett, R. A. Keller, “Large DNA fragment sizing by flow cytometry: application to the characterization of P1 artificial chromosome (PAC) clones,” Nucleic Acids Res. 24, 4202–4209 (1996).
[CrossRef] [PubMed]

1995 (4)

A. Castro, E. B. Shera, “Single molecule electrophoresis,” Anal. Chem. 67, 3181–3186 (1995).
[CrossRef]

J. T. Petty, M. E. Johnson, P. M. Goodwin, J. C. Martin, J. H. Jett, R. A. Keller, “Characterization of DNA size determination of small fragments by flow cytometry,” Anal. Chem. 67, 1755–1761 (1995).
[CrossRef]

J. P. Jacobsen, J. B. Pedersen, L. F. Hansen, D. E. Wemmer, “Site selective bis-intercalation of a homodimeric thiazole orange dye in DNA oligonucleotides,” Nucleic Acids Res. 23, 753–760 (1995).
[CrossRef] [PubMed]

H. P. Spielmann, D. E. Wemmer, J. Jacobsen, “Solution structure of a DNA complex with the fluorescent bis-intercalator TOTO determined by NMR spectroscopy,” Biochemistry 34, 8542–8553 (1995).
[CrossRef] [PubMed]

1994 (1)

A. Larsson, C. Carlsson, M. Jonsson, B. Albinsson, “Characterization of the binding of the fluorescent dyes YO and YOYO to DNA by polarized light spectroscopy,” J. Am. Chem. Soc. 116, 8459–8465 (1994).
[CrossRef]

1993 (2)

P. M. Goodwin, M. E. Johnson, J. C. Martin, W. P. Ambrose, B. L. Marrone, J. H. Jett, R. A. Keller, “Rapid sizing of individual fluorescently stained DNA fragments by flow cytometry,” Nucleic Acids Res. 21, 803–806 (1993).
[CrossRef] [PubMed]

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

1979 (1)

D. Axelrod, “Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization,” Biophys. J. 26, 557–574 (1979).
[CrossRef] [PubMed]

1975 (1)

C. C. Tsai, S. C. Jain, H. M. Sobell, “X-ray crystallographic visualization of drug-nucleic acid intercalative binding: structure of an ethidium-dinucleoside monophosphate crystalline complex, ethidium: 5-iodouridylyl(3′-5′) adenosine,” Proc. Natl. Acad. Sci. USA 72, 628–632 (1975).
[CrossRef]

Agronskaia, A.

A. Agronskaia, J. M. Schins, B. G. de Grooth, J. Greve, “Polarization effects in flow cytometric DNA sizing,” Appl. Opt. 38, 714–719 (1999).
[CrossRef]

A. Agronskaia, A. Florians, K. O. van der Werf, J. M. Schins, B. G. de Grooth, J. Greve, “Photon-counting device compatible with conventional flow cytometric data acquisition electronics,” Cytometry 32, 255–259 (1998).
[CrossRef] [PubMed]

J. M. Schins, A. Agronskaia, B. G. de Grooth, J. Greve, “New technique for high resolution DNA sizing in epi-illumination,” Cytometry 32, 132–136 (1998).
[CrossRef] [PubMed]

Albinsson, B.

A. Larsson, C. Carlsson, M. Jonsson, B. Albinsson, “Characterization of the binding of the fluorescent dyes YO and YOYO to DNA by polarized light spectroscopy,” J. Am. Chem. Soc. 116, 8459–8465 (1994).
[CrossRef]

Al-binsson, B.

C. Carlsson, A. Larsson, M. Bjorkman, M. Jonsson, B. Al-binsson, “Experimental and simulated fluorescence depolarization due to energy transfer as tools to study DNA-dye interactions,” Biopolymers 41, 481–494 (1997).
[CrossRef]

Ambrose, W. P.

J. Enderlein, D. L. Robbins, W. P. Ambrose, R. A. Keller, “Molecular shot noise, burst size distribution, and single-molecule detection in fluid flow: effects of multiple occupancy,” J. Phys. Chem. 102, 6089–6094 (1998).
[CrossRef]

P. M. Goodwin, M. E. Johnson, J. C. Martin, W. P. Ambrose, B. L. Marrone, J. H. Jett, R. A. Keller, “Rapid sizing of individual fluorescently stained DNA fragments by flow cytometry,” Nucleic Acids Res. 21, 803–806 (1993).
[CrossRef] [PubMed]

W. P. Ambrose, H. Cai, P. M. Goodwin, J. H. Jett, R. C. Habbersett, E. J. Larson, W. K. Grace, J. H. Werner, R. A. Keller, “Flow cytometric sizing of DNA fragments,” in Topics in Fluorescence: DNA Technology, J. R. Lacowicz, ed. (Plenum, New York, 2000), Vol. 7.

M. E. Johnson, P. M. Goodwin, W. P. Ambrose, J. C. Martin, B. L. Marrone, J. H. Jett, R. A. Keller, “Sizing of DNA fragments by flow cytometry,” in Advances in FluorescenceSensing Technology IV, J. R. Lakowicz, S. A. Soper, R. B. Thompson, eds., Proc. SPIE1895, 69–78 (1993).

Axelrod, D.

D. Axelrod, “Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization,” Biophys. J. 26, 557–574 (1979).
[CrossRef] [PubMed]

D. Axelrod, “Fluorescence polarization microscopy,” in Methods in Cell Biology (Academic, San Diego, Calif., 1989), Vol. 30, pp. 333–352.
[CrossRef]

Babcock, H. P.

D. E. Smith, H. P. Babcock, S. Chu, “Single polymer dynamics in steady shear flow,” Science 283, 1724–1727 (1999).
[CrossRef] [PubMed]

Bjorkman, M.

C. Carlsson, A. Larsson, M. Bjorkman, M. Jonsson, B. Al-binsson, “Experimental and simulated fluorescence depolarization due to energy transfer as tools to study DNA-dye interactions,” Biopolymers 41, 481–494 (1997).
[CrossRef]

Brown, N. C.

Z. Huang, J. T. Petty, B. O’Quinn, J. L. Longmire, N. C. Brown, J. H. Jett, R. A. Keller, “Large DNA fragment sizing by flow cytometry: application to the characterization of P1 artificial chromosome (PAC) clones,” Nucleic Acids Res. 24, 4202–4209 (1996).
[CrossRef] [PubMed]

Cai, H.

A. Van Orden, H. Cai, P. M. Goodwin, 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]

W. P. Ambrose, H. Cai, P. M. Goodwin, J. H. Jett, R. C. Habbersett, E. J. Larson, W. K. Grace, J. H. Werner, R. A. Keller, “Flow cytometric sizing of DNA fragments,” in Topics in Fluorescence: DNA Technology, J. R. Lacowicz, ed. (Plenum, New York, 2000), Vol. 7.

Carlsson, C.

C. Carlsson, A. Larsson, M. Bjorkman, M. Jonsson, B. Al-binsson, “Experimental and simulated fluorescence depolarization due to energy transfer as tools to study DNA-dye interactions,” Biopolymers 41, 481–494 (1997).
[CrossRef]

A. Larsson, C. Carlsson, M. Jonsson, B. Albinsson, “Characterization of the binding of the fluorescent dyes YO and YOYO to DNA by polarized light spectroscopy,” J. Am. Chem. Soc. 116, 8459–8465 (1994).
[CrossRef]

Castro, A.

A. Castro, E. B. Shera, “Single molecule electrophoresis,” Anal. Chem. 67, 3181–3186 (1995).
[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]

Chou, H. P.

H. P. Chou, C. Spence, A. Scherer, S. Quake, “A microfabricated device for sizing and sorting DNA molecules,” Proc. Natl. Acad. Sci. USA 96, 11–13 (1999).
[CrossRef] [PubMed]

Chu, S.

D. E. Smith, H. P. Babcock, S. Chu, “Single polymer dynamics in steady shear flow,” Science 283, 1724–1727 (1999).
[CrossRef] [PubMed]

T. T. Perkins, D. E. Smith, S. Chu, “Single polymer dynamics in an elongational flow,” Science 276, 2016–2021 (1997).
[CrossRef] [PubMed]

de Grooth, B. G.

A. Agronskaia, J. M. Schins, B. G. de Grooth, J. Greve, “Polarization effects in flow cytometric DNA sizing,” Appl. Opt. 38, 714–719 (1999).
[CrossRef]

A. Agronskaia, A. Florians, K. O. van der Werf, J. M. Schins, B. G. de Grooth, J. Greve, “Photon-counting device compatible with conventional flow cytometric data acquisition electronics,” Cytometry 32, 255–259 (1998).
[CrossRef] [PubMed]

J. M. Schins, A. Agronskaia, B. G. de Grooth, J. Greve, “New technique for high resolution DNA sizing in epi-illumination,” Cytometry 32, 132–136 (1998).
[CrossRef] [PubMed]

Enderlein, J.

J. Enderlein, D. L. Robbins, W. P. Ambrose, R. A. Keller, “Molecular shot noise, burst size distribution, and single-molecule detection in fluid flow: effects of multiple occupancy,” J. Phys. Chem. 102, 6089–6094 (1998).
[CrossRef]

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]

Florians, A.

A. Agronskaia, A. Florians, K. O. van der Werf, J. M. Schins, B. G. de Grooth, J. Greve, “Photon-counting device compatible with conventional flow cytometric data acquisition electronics,” Cytometry 32, 255–259 (1998).
[CrossRef] [PubMed]

Goodwin, P. M.

A. Van Orden, H. Cai, P. M. Goodwin, 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]

J. T. Petty, M. E. Johnson, P. M. Goodwin, J. C. Martin, J. H. Jett, R. A. Keller, “Characterization of DNA size determination of small fragments by flow cytometry,” Anal. Chem. 67, 1755–1761 (1995).
[CrossRef]

P. M. Goodwin, M. E. Johnson, J. C. Martin, W. P. Ambrose, B. L. Marrone, J. H. Jett, R. A. Keller, “Rapid sizing of individual fluorescently stained DNA fragments by flow cytometry,” Nucleic Acids Res. 21, 803–806 (1993).
[CrossRef] [PubMed]

W. P. Ambrose, H. Cai, P. M. Goodwin, J. H. Jett, R. C. Habbersett, E. J. Larson, W. K. Grace, J. H. Werner, R. A. Keller, “Flow cytometric sizing of DNA fragments,” in Topics in Fluorescence: DNA Technology, J. R. Lacowicz, ed. (Plenum, New York, 2000), Vol. 7.

M. E. Johnson, P. M. Goodwin, W. P. Ambrose, J. C. Martin, B. L. Marrone, J. H. Jett, R. A. Keller, “Sizing of DNA fragments by flow cytometry,” in Advances in FluorescenceSensing Technology IV, J. R. Lakowicz, S. A. Soper, R. B. Thompson, eds., Proc. SPIE1895, 69–78 (1993).

Grace, W. K.

W. P. Ambrose, H. Cai, P. M. Goodwin, J. H. Jett, R. C. Habbersett, E. J. Larson, W. K. Grace, J. H. Werner, R. A. Keller, “Flow cytometric sizing of DNA fragments,” in Topics in Fluorescence: DNA Technology, J. R. Lacowicz, ed. (Plenum, New York, 2000), Vol. 7.

Greve, J.

A. Agronskaia, J. M. Schins, B. G. de Grooth, J. Greve, “Polarization effects in flow cytometric DNA sizing,” Appl. Opt. 38, 714–719 (1999).
[CrossRef]

A. Agronskaia, A. Florians, K. O. van der Werf, J. M. Schins, B. G. de Grooth, J. Greve, “Photon-counting device compatible with conventional flow cytometric data acquisition electronics,” Cytometry 32, 255–259 (1998).
[CrossRef] [PubMed]

J. M. Schins, A. Agronskaia, B. G. de Grooth, J. Greve, “New technique for high resolution DNA sizing in epi-illumination,” Cytometry 32, 132–136 (1998).
[CrossRef] [PubMed]

Habbersett, R. C.

W. P. Ambrose, H. Cai, P. M. Goodwin, J. H. Jett, R. C. Habbersett, E. J. Larson, W. K. Grace, J. H. Werner, R. A. Keller, “Flow cytometric sizing of DNA fragments,” in Topics in Fluorescence: DNA Technology, J. R. Lacowicz, ed. (Plenum, New York, 2000), Vol. 7.

Hansen, L. F.

J. P. Jacobsen, J. B. Pedersen, L. F. Hansen, D. E. Wemmer, “Site selective bis-intercalation of a homodimeric thiazole orange dye in DNA oligonucleotides,” Nucleic Acids Res. 23, 753–760 (1995).
[CrossRef] [PubMed]

Huang, Z.

Z. Huang, J. H. Jett, R. A. Keller, “Bacteria genome fingerprinting by flow cytometry,” Cytometry 35, 169–175 (1999).
[CrossRef] [PubMed]

Z. Huang, J. T. Petty, B. O’Quinn, J. L. Longmire, N. C. Brown, J. H. Jett, R. A. Keller, “Large DNA fragment sizing by flow cytometry: application to the characterization of P1 artificial chromosome (PAC) clones,” Nucleic Acids Res. 24, 4202–4209 (1996).
[CrossRef] [PubMed]

Jackson, J. D.

J. D. Jackson, Classical Electrodynamics, (Wiley, New York, 1975), p. 138.

Jacobsen, J.

H. P. Spielmann, D. E. Wemmer, J. Jacobsen, “Solution structure of a DNA complex with the fluorescent bis-intercalator TOTO determined by NMR spectroscopy,” Biochemistry 34, 8542–8553 (1995).
[CrossRef] [PubMed]

Jacobsen, J. P.

J. P. Jacobsen, J. B. Pedersen, L. F. Hansen, D. E. Wemmer, “Site selective bis-intercalation of a homodimeric thiazole orange dye in DNA oligonucleotides,” Nucleic Acids Res. 23, 753–760 (1995).
[CrossRef] [PubMed]

Jain, S. C.

C. C. Tsai, S. C. Jain, H. M. Sobell, “X-ray crystallographic visualization of drug-nucleic acid intercalative binding: structure of an ethidium-dinucleoside monophosphate crystalline complex, ethidium: 5-iodouridylyl(3′-5′) adenosine,” Proc. Natl. Acad. Sci. USA 72, 628–632 (1975).
[CrossRef]

Jett, J. H.

Y. Kim, J. H. Jett, E. J. Larson, J. R. Penttila, B. L. Marrone, R. A. Keller, “Bacterial fingerprinting by flow cytometry: bacterial species discrimination,” Cytometry 36, 1–9 (1999).
[CrossRef]

Z. Huang, J. H. Jett, R. A. Keller, “Bacteria genome fingerprinting by flow cytometry,” Cytometry 35, 169–175 (1999).
[CrossRef] [PubMed]

Z. Huang, J. T. Petty, B. O’Quinn, J. L. Longmire, N. C. Brown, J. H. Jett, R. A. Keller, “Large DNA fragment sizing by flow cytometry: application to the characterization of P1 artificial chromosome (PAC) clones,” Nucleic Acids Res. 24, 4202–4209 (1996).
[CrossRef] [PubMed]

J. T. Petty, M. E. Johnson, P. M. Goodwin, J. C. Martin, J. H. Jett, R. A. Keller, “Characterization of DNA size determination of small fragments by flow cytometry,” Anal. Chem. 67, 1755–1761 (1995).
[CrossRef]

P. M. Goodwin, M. E. Johnson, J. C. Martin, W. P. Ambrose, B. L. Marrone, J. H. Jett, R. A. Keller, “Rapid sizing of individual fluorescently stained DNA fragments by flow cytometry,” Nucleic Acids Res. 21, 803–806 (1993).
[CrossRef] [PubMed]

W. P. Ambrose, H. Cai, P. M. Goodwin, J. H. Jett, R. C. Habbersett, E. J. Larson, W. K. Grace, J. H. Werner, R. A. Keller, “Flow cytometric sizing of DNA fragments,” in Topics in Fluorescence: DNA Technology, J. R. Lacowicz, ed. (Plenum, New York, 2000), Vol. 7.

M. E. Johnson, P. M. Goodwin, W. P. Ambrose, J. C. Martin, B. L. Marrone, J. H. Jett, R. A. Keller, “Sizing of DNA fragments by flow cytometry,” in Advances in FluorescenceSensing Technology IV, J. R. Lakowicz, S. A. Soper, R. B. Thompson, eds., Proc. SPIE1895, 69–78 (1993).

Johnson, M. E.

J. T. Petty, M. E. Johnson, P. M. Goodwin, J. C. Martin, J. H. Jett, R. A. Keller, “Characterization of DNA size determination of small fragments by flow cytometry,” Anal. Chem. 67, 1755–1761 (1995).
[CrossRef]

P. M. Goodwin, M. E. Johnson, J. C. Martin, W. P. Ambrose, B. L. Marrone, J. H. Jett, R. A. Keller, “Rapid sizing of individual fluorescently stained DNA fragments by flow cytometry,” Nucleic Acids Res. 21, 803–806 (1993).
[CrossRef] [PubMed]

M. E. Johnson, P. M. Goodwin, W. P. Ambrose, J. C. Martin, B. L. Marrone, J. H. Jett, R. A. Keller, “Sizing of DNA fragments by flow cytometry,” in Advances in FluorescenceSensing Technology IV, J. R. Lakowicz, S. A. Soper, R. B. Thompson, eds., Proc. SPIE1895, 69–78 (1993).

Jonsson, M.

C. Carlsson, A. Larsson, M. Bjorkman, M. Jonsson, B. Al-binsson, “Experimental and simulated fluorescence depolarization due to energy transfer as tools to study DNA-dye interactions,” Biopolymers 41, 481–494 (1997).
[CrossRef]

A. Larsson, C. Carlsson, M. Jonsson, B. Albinsson, “Characterization of the binding of the fluorescent dyes YO and YOYO to DNA by polarized light spectroscopy,” J. Am. Chem. Soc. 116, 8459–8465 (1994).
[CrossRef]

Keller, R. A.

Y. Kim, J. H. Jett, E. J. Larson, J. R. Penttila, B. L. Marrone, R. A. Keller, “Bacterial fingerprinting by flow cytometry: bacterial species discrimination,” Cytometry 36, 1–9 (1999).
[CrossRef]

Z. Huang, J. H. Jett, R. A. Keller, “Bacteria genome fingerprinting by flow cytometry,” Cytometry 35, 169–175 (1999).
[CrossRef] [PubMed]

A. Van Orden, H. Cai, P. M. Goodwin, 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]

J. Enderlein, D. L. Robbins, W. P. Ambrose, R. A. Keller, “Molecular shot noise, burst size distribution, and single-molecule detection in fluid flow: effects of multiple occupancy,” J. Phys. Chem. 102, 6089–6094 (1998).
[CrossRef]

Z. Huang, J. T. Petty, B. O’Quinn, J. L. Longmire, N. C. Brown, J. H. Jett, R. A. Keller, “Large DNA fragment sizing by flow cytometry: application to the characterization of P1 artificial chromosome (PAC) clones,” Nucleic Acids Res. 24, 4202–4209 (1996).
[CrossRef] [PubMed]

J. T. Petty, M. E. Johnson, P. M. Goodwin, J. C. Martin, J. H. Jett, R. A. Keller, “Characterization of DNA size determination of small fragments by flow cytometry,” Anal. Chem. 67, 1755–1761 (1995).
[CrossRef]

P. M. Goodwin, M. E. Johnson, J. C. Martin, W. P. Ambrose, B. L. Marrone, J. H. Jett, R. A. Keller, “Rapid sizing of individual fluorescently stained DNA fragments by flow cytometry,” Nucleic Acids Res. 21, 803–806 (1993).
[CrossRef] [PubMed]

W. P. Ambrose, H. Cai, P. M. Goodwin, J. H. Jett, R. C. Habbersett, E. J. Larson, W. K. Grace, J. H. Werner, R. A. Keller, “Flow cytometric sizing of DNA fragments,” in Topics in Fluorescence: DNA Technology, J. R. Lacowicz, ed. (Plenum, New York, 2000), Vol. 7.

M. E. Johnson, P. M. Goodwin, W. P. Ambrose, J. C. Martin, B. L. Marrone, J. H. Jett, R. A. Keller, “Sizing of DNA fragments by flow cytometry,” in Advances in FluorescenceSensing Technology IV, J. R. Lakowicz, S. A. Soper, R. B. Thompson, eds., Proc. SPIE1895, 69–78 (1993).

Kim, Y.

Y. Kim, J. H. Jett, E. J. Larson, J. R. Penttila, B. L. Marrone, R. A. Keller, “Bacterial fingerprinting by flow cytometry: bacterial species discrimination,” Cytometry 36, 1–9 (1999).
[CrossRef]

Larson, E. J.

Y. Kim, J. H. Jett, E. J. Larson, J. R. Penttila, B. L. Marrone, R. A. Keller, “Bacterial fingerprinting by flow cytometry: bacterial species discrimination,” Cytometry 36, 1–9 (1999).
[CrossRef]

W. P. Ambrose, H. Cai, P. M. Goodwin, J. H. Jett, R. C. Habbersett, E. J. Larson, W. K. Grace, J. H. Werner, R. A. Keller, “Flow cytometric sizing of DNA fragments,” in Topics in Fluorescence: DNA Technology, J. R. Lacowicz, ed. (Plenum, New York, 2000), Vol. 7.

Larsson, A.

C. Carlsson, A. Larsson, M. Bjorkman, M. Jonsson, B. Al-binsson, “Experimental and simulated fluorescence depolarization due to energy transfer as tools to study DNA-dye interactions,” Biopolymers 41, 481–494 (1997).
[CrossRef]

A. Larsson, C. Carlsson, M. Jonsson, B. Albinsson, “Characterization of the binding of the fluorescent dyes YO and YOYO to DNA by polarized light spectroscopy,” J. Am. Chem. Soc. 116, 8459–8465 (1994).
[CrossRef]

Longmire, J. L.

Z. Huang, J. T. Petty, B. O’Quinn, J. L. Longmire, N. C. Brown, J. H. Jett, R. A. Keller, “Large DNA fragment sizing by flow cytometry: application to the characterization of P1 artificial chromosome (PAC) clones,” Nucleic Acids Res. 24, 4202–4209 (1996).
[CrossRef] [PubMed]

Marrone, B. L.

Y. Kim, J. H. Jett, E. J. Larson, J. R. Penttila, B. L. Marrone, R. A. Keller, “Bacterial fingerprinting by flow cytometry: bacterial species discrimination,” Cytometry 36, 1–9 (1999).
[CrossRef]

P. M. Goodwin, M. E. Johnson, J. C. Martin, W. P. Ambrose, B. L. Marrone, J. H. Jett, R. A. Keller, “Rapid sizing of individual fluorescently stained DNA fragments by flow cytometry,” Nucleic Acids Res. 21, 803–806 (1993).
[CrossRef] [PubMed]

M. E. Johnson, P. M. Goodwin, W. P. Ambrose, J. C. Martin, B. L. Marrone, J. H. Jett, R. A. Keller, “Sizing of DNA fragments by flow cytometry,” in Advances in FluorescenceSensing Technology IV, J. R. Lakowicz, S. A. Soper, R. B. Thompson, eds., Proc. SPIE1895, 69–78 (1993).

Martin, J. C.

J. T. Petty, M. E. Johnson, P. M. Goodwin, J. C. Martin, J. H. Jett, R. A. Keller, “Characterization of DNA size determination of small fragments by flow cytometry,” Anal. Chem. 67, 1755–1761 (1995).
[CrossRef]

P. M. Goodwin, M. E. Johnson, J. C. Martin, W. P. Ambrose, B. L. Marrone, J. H. Jett, R. A. Keller, “Rapid sizing of individual fluorescently stained DNA fragments by flow cytometry,” Nucleic Acids Res. 21, 803–806 (1993).
[CrossRef] [PubMed]

M. E. Johnson, P. M. Goodwin, W. P. Ambrose, J. C. Martin, B. L. Marrone, J. H. Jett, R. A. Keller, “Sizing of DNA fragments by flow cytometry,” in Advances in FluorescenceSensing Technology IV, J. R. Lakowicz, S. A. Soper, R. B. Thompson, eds., Proc. SPIE1895, 69–78 (1993).

O’Quinn, B.

Z. Huang, J. T. Petty, B. O’Quinn, J. L. Longmire, N. C. Brown, J. H. Jett, R. A. Keller, “Large DNA fragment sizing by flow cytometry: application to the characterization of P1 artificial chromosome (PAC) clones,” Nucleic Acids Res. 24, 4202–4209 (1996).
[CrossRef] [PubMed]

Pedersen, J. B.

J. P. Jacobsen, J. B. Pedersen, L. F. Hansen, D. E. Wemmer, “Site selective bis-intercalation of a homodimeric thiazole orange dye in DNA oligonucleotides,” Nucleic Acids Res. 23, 753–760 (1995).
[CrossRef] [PubMed]

Penttila, J. R.

Y. Kim, J. H. Jett, E. J. Larson, J. R. Penttila, B. L. Marrone, R. A. Keller, “Bacterial fingerprinting by flow cytometry: bacterial species discrimination,” Cytometry 36, 1–9 (1999).
[CrossRef]

Perkins, T. T.

T. T. Perkins, D. E. Smith, S. Chu, “Single polymer dynamics in an elongational flow,” Science 276, 2016–2021 (1997).
[CrossRef] [PubMed]

Petty, J. T.

Z. Huang, J. T. Petty, B. O’Quinn, J. L. Longmire, N. C. Brown, J. H. Jett, R. A. Keller, “Large DNA fragment sizing by flow cytometry: application to the characterization of P1 artificial chromosome (PAC) clones,” Nucleic Acids Res. 24, 4202–4209 (1996).
[CrossRef] [PubMed]

J. T. Petty, M. E. Johnson, P. M. Goodwin, J. C. Martin, J. H. Jett, R. A. Keller, “Characterization of DNA size determination of small fragments by flow cytometry,” Anal. Chem. 67, 1755–1761 (1995).
[CrossRef]

Quake, S.

H. P. Chou, C. Spence, A. Scherer, S. Quake, “A microfabricated device for sizing and sorting DNA molecules,” Proc. Natl. Acad. Sci. USA 96, 11–13 (1999).
[CrossRef] [PubMed]

Robbins, D. L.

J. Enderlein, D. L. Robbins, W. P. Ambrose, R. A. Keller, “Molecular shot noise, burst size distribution, and single-molecule detection in fluid flow: effects of multiple occupancy,” J. Phys. Chem. 102, 6089–6094 (1998).
[CrossRef]

Scherer, A.

H. P. Chou, C. Spence, A. Scherer, S. Quake, “A microfabricated device for sizing and sorting DNA molecules,” Proc. Natl. Acad. Sci. USA 96, 11–13 (1999).
[CrossRef] [PubMed]

Schins, J. M.

A. Agronskaia, J. M. Schins, B. G. de Grooth, J. Greve, “Polarization effects in flow cytometric DNA sizing,” Appl. Opt. 38, 714–719 (1999).
[CrossRef]

A. Agronskaia, A. Florians, K. O. van der Werf, J. M. Schins, B. G. de Grooth, J. Greve, “Photon-counting device compatible with conventional flow cytometric data acquisition electronics,” Cytometry 32, 255–259 (1998).
[CrossRef] [PubMed]

J. M. Schins, A. Agronskaia, B. G. de Grooth, J. Greve, “New technique for high resolution DNA sizing in epi-illumination,” Cytometry 32, 132–136 (1998).
[CrossRef] [PubMed]

Shera, E. B.

A. Castro, E. B. Shera, “Single molecule electrophoresis,” Anal. Chem. 67, 3181–3186 (1995).
[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]

Smith, D. E.

D. E. Smith, H. P. Babcock, S. Chu, “Single polymer dynamics in steady shear flow,” Science 283, 1724–1727 (1999).
[CrossRef] [PubMed]

T. T. Perkins, D. E. Smith, S. Chu, “Single polymer dynamics in an elongational flow,” Science 276, 2016–2021 (1997).
[CrossRef] [PubMed]

Sobell, H. M.

C. C. Tsai, S. C. Jain, H. M. Sobell, “X-ray crystallographic visualization of drug-nucleic acid intercalative binding: structure of an ethidium-dinucleoside monophosphate crystalline complex, ethidium: 5-iodouridylyl(3′-5′) adenosine,” Proc. Natl. Acad. Sci. USA 72, 628–632 (1975).
[CrossRef]

Spence, C.

H. P. Chou, C. Spence, A. Scherer, S. Quake, “A microfabricated device for sizing and sorting DNA molecules,” Proc. Natl. Acad. Sci. USA 96, 11–13 (1999).
[CrossRef] [PubMed]

Spielmann, H. P.

H. P. Spielmann, D. E. Wemmer, J. Jacobsen, “Solution structure of a DNA complex with the fluorescent bis-intercalator TOTO determined by NMR spectroscopy,” Biochemistry 34, 8542–8553 (1995).
[CrossRef] [PubMed]

Tsai, C. C.

C. C. Tsai, S. C. Jain, H. M. Sobell, “X-ray crystallographic visualization of drug-nucleic acid intercalative binding: structure of an ethidium-dinucleoside monophosphate crystalline complex, ethidium: 5-iodouridylyl(3′-5′) adenosine,” Proc. Natl. Acad. Sci. USA 72, 628–632 (1975).
[CrossRef]

van der Werf, K. O.

A. Agronskaia, A. Florians, K. O. van der Werf, J. M. Schins, B. G. de Grooth, J. Greve, “Photon-counting device compatible with conventional flow cytometric data acquisition electronics,” Cytometry 32, 255–259 (1998).
[CrossRef] [PubMed]

Van Orden, A.

A. Van Orden, H. Cai, P. M. Goodwin, 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]

Wemmer, D. E.

J. P. Jacobsen, J. B. Pedersen, L. F. Hansen, D. E. Wemmer, “Site selective bis-intercalation of a homodimeric thiazole orange dye in DNA oligonucleotides,” Nucleic Acids Res. 23, 753–760 (1995).
[CrossRef] [PubMed]

H. P. Spielmann, D. E. Wemmer, J. Jacobsen, “Solution structure of a DNA complex with the fluorescent bis-intercalator TOTO determined by NMR spectroscopy,” Biochemistry 34, 8542–8553 (1995).
[CrossRef] [PubMed]

Werner, J. H.

W. P. Ambrose, H. Cai, P. M. Goodwin, J. H. Jett, R. C. Habbersett, E. J. Larson, W. K. Grace, J. H. Werner, R. A. Keller, “Flow cytometric sizing of DNA fragments,” in Topics in Fluorescence: DNA Technology, J. R. Lacowicz, ed. (Plenum, New York, 2000), Vol. 7.

Anal. Chem. (4)

J. T. Petty, M. E. Johnson, P. M. Goodwin, J. C. Martin, J. H. Jett, R. A. Keller, “Characterization of DNA size determination of small fragments by flow cytometry,” Anal. Chem. 67, 1755–1761 (1995).
[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]

A. Castro, E. B. Shera, “Single molecule electrophoresis,” Anal. Chem. 67, 3181–3186 (1995).
[CrossRef]

A. Van Orden, H. Cai, P. M. Goodwin, 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]

Appl. Opt. (1)

Biochemistry (1)

H. P. Spielmann, D. E. Wemmer, J. Jacobsen, “Solution structure of a DNA complex with the fluorescent bis-intercalator TOTO determined by NMR spectroscopy,” Biochemistry 34, 8542–8553 (1995).
[CrossRef] [PubMed]

Biophys. J. (1)

D. Axelrod, “Carbocyanine dye orientation in red cell membrane studied by microscopic fluorescence polarization,” Biophys. J. 26, 557–574 (1979).
[CrossRef] [PubMed]

Biopolymers (1)

C. Carlsson, A. Larsson, M. Bjorkman, M. Jonsson, B. Al-binsson, “Experimental and simulated fluorescence depolarization due to energy transfer as tools to study DNA-dye interactions,” Biopolymers 41, 481–494 (1997).
[CrossRef]

Cytometry (4)

Z. Huang, J. H. Jett, R. A. Keller, “Bacteria genome fingerprinting by flow cytometry,” Cytometry 35, 169–175 (1999).
[CrossRef] [PubMed]

Y. Kim, J. H. Jett, E. J. Larson, J. R. Penttila, B. L. Marrone, R. A. Keller, “Bacterial fingerprinting by flow cytometry: bacterial species discrimination,” Cytometry 36, 1–9 (1999).
[CrossRef]

J. M. Schins, A. Agronskaia, B. G. de Grooth, J. Greve, “New technique for high resolution DNA sizing in epi-illumination,” Cytometry 32, 132–136 (1998).
[CrossRef] [PubMed]

A. Agronskaia, A. Florians, K. O. van der Werf, J. M. Schins, B. G. de Grooth, J. Greve, “Photon-counting device compatible with conventional flow cytometric data acquisition electronics,” Cytometry 32, 255–259 (1998).
[CrossRef] [PubMed]

J. Am. Chem. Soc. (1)

A. Larsson, C. Carlsson, M. Jonsson, B. Albinsson, “Characterization of the binding of the fluorescent dyes YO and YOYO to DNA by polarized light spectroscopy,” J. Am. Chem. Soc. 116, 8459–8465 (1994).
[CrossRef]

J. Phys. Chem. (1)

J. Enderlein, D. L. Robbins, W. P. Ambrose, R. A. Keller, “Molecular shot noise, burst size distribution, and single-molecule detection in fluid flow: effects of multiple occupancy,” J. Phys. Chem. 102, 6089–6094 (1998).
[CrossRef]

Nucleic Acids Res. (3)

Z. Huang, J. T. Petty, B. O’Quinn, J. L. Longmire, N. C. Brown, J. H. Jett, R. A. Keller, “Large DNA fragment sizing by flow cytometry: application to the characterization of P1 artificial chromosome (PAC) clones,” Nucleic Acids Res. 24, 4202–4209 (1996).
[CrossRef] [PubMed]

P. M. Goodwin, M. E. Johnson, J. C. Martin, W. P. Ambrose, B. L. Marrone, J. H. Jett, R. A. Keller, “Rapid sizing of individual fluorescently stained DNA fragments by flow cytometry,” Nucleic Acids Res. 21, 803–806 (1993).
[CrossRef] [PubMed]

J. P. Jacobsen, J. B. Pedersen, L. F. Hansen, D. E. Wemmer, “Site selective bis-intercalation of a homodimeric thiazole orange dye in DNA oligonucleotides,” Nucleic Acids Res. 23, 753–760 (1995).
[CrossRef] [PubMed]

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

H. P. Chou, C. Spence, A. Scherer, S. Quake, “A microfabricated device for sizing and sorting DNA molecules,” Proc. Natl. Acad. Sci. USA 96, 11–13 (1999).
[CrossRef] [PubMed]

C. C. Tsai, S. C. Jain, H. M. Sobell, “X-ray crystallographic visualization of drug-nucleic acid intercalative binding: structure of an ethidium-dinucleoside monophosphate crystalline complex, ethidium: 5-iodouridylyl(3′-5′) adenosine,” Proc. Natl. Acad. Sci. USA 72, 628–632 (1975).
[CrossRef]

Science (2)

T. T. Perkins, D. E. Smith, S. Chu, “Single polymer dynamics in an elongational flow,” Science 276, 2016–2021 (1997).
[CrossRef] [PubMed]

D. E. Smith, H. P. Babcock, S. Chu, “Single polymer dynamics in steady shear flow,” Science 283, 1724–1727 (1999).
[CrossRef] [PubMed]

Other (4)

J. D. Jackson, Classical Electrodynamics, (Wiley, New York, 1975), p. 138.

W. P. Ambrose, H. Cai, P. M. Goodwin, J. H. Jett, R. C. Habbersett, E. J. Larson, W. K. Grace, J. H. Werner, R. A. Keller, “Flow cytometric sizing of DNA fragments,” in Topics in Fluorescence: DNA Technology, J. R. Lacowicz, ed. (Plenum, New York, 2000), Vol. 7.

M. E. Johnson, P. M. Goodwin, W. P. Ambrose, J. C. Martin, B. L. Marrone, J. H. Jett, R. A. Keller, “Sizing of DNA fragments by flow cytometry,” in Advances in FluorescenceSensing Technology IV, J. R. Lakowicz, S. A. Soper, R. B. Thompson, eds., Proc. SPIE1895, 69–78 (1993).

D. Axelrod, “Fluorescence polarization microscopy,” in Methods in Cell Biology (Academic, San Diego, Calif., 1989), Vol. 30, pp. 333–352.
[CrossRef]

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Figures (6)

Fig. 1
Fig. 1

Cartesian diagram of the flow system and optical setup. Flow is in the - direction, the excitation laser propagates along the ŷ direction and is polarized in the direction in the diagram (90° with respect to the flow axis). Fluorescence is collected along the x axis. A dipole moment p was placed at the origin of the diagram. The polarization angle of the excitation laser was varied in this research from the 90° depicted to three other polarization angles (all in the xz plane) (see text, Subsection 3.E). In epiexcitation, the excitation laser counterpropagates down the optical collection (x) axis, and polarizations only in the yz plane are accessible.

Fig. 2
Fig. 2

Burst size histogram and fit. Data were taken with excitation laser polarization parallel to the x axis of Fig. 1. The fragment lengths of the observed peaks are 4.36, 6.55, 9.42, 23.1, 48.5, and 97.0 kbp.

Fig. 3
Fig. 3

(A) Centroids extracted from Fig. 2 versus known fragment length with a linear fit to the first three data points extrapolated over the entire data range. The excitation laser polarization was oriented parallel to the x axis. DNA fragment sizes (kbp) and burst sizes [photoelectrons (pe)] are (4.36, 176), (6.55, 282), (9.42, 396), (23.1, 1066), (48.5, 2193), and (97.0, 4340). (B) Burst sizes versus fragment lengths taken when the excitation laser polarization was oriented parallel to the z axis. A linear fit to the smallest three DNA fragments was extrapolated over the entire data range, as in (A). The DNA fragment sizes (kbp) and burst sizes (pe) are (4.36, 297), (6.55, 426), (9.42, 601), (23.1, 1351), (48.5, 2374), and (97.0, 4030). The ND 0.3 filter was removed to yield similar signal levels to the data presented in (A).

Fig. 4
Fig. 4

Fit residuals (expressed as a percent deviation) for the data presented in Fig. 3 for excitation polarization parallel to the x axis (open circles) and the z axis (filled squares). The residuals for excitation polarization parallel to the z axis are larger than the residuals to the data taken with the excitation polarization parallel to the x axis. Moreover, for excitation polarization parallel to the z axis, the residuals to the fit show deviations that increase with fragment length.

Fig. 5
Fig. 5

(A) Normalized autocorrelation functions and theoretical fits for 48.5 DNA at three different flow velocities (1.1, 2.4, and 6.6 cm/s). (B) Normalized autocorrelation functions and theoretical fits for 1.8-kbp DNA at the same flow velocities. The longer correlation times for 48.5 DNA are attributed to alignment and elongation of these fragments by the flow stream. For these measurements, a circular 11-µm, 1/e 2 diameter spot was used to enhance the spatial resolution.

Fig. 6
Fig. 6

(A) Anticipated fluorescence intensity for three different excitation polarizations as a function of the angle between the dipole moment axis and the flow axis. (B) Fluorescence intensity expected for optimal excitation polarization angle for epiexcitation (57.4°) and orthogonal excitation (68°) geometries.

Tables (2)

Tables Icon

Table 1 Measured Burst Sizes

Tables Icon

Table 2 CV for 48.5-kbp DNA over a Range of Excitation Angles

Equations (2)

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

Fxx=38 sin4θ,  Fyx=18 sin4θ,  Fzx=12 sin2θcos2θ,  Fxy=18 sin4θ,  Fyy=38 sin4θ,  Fzy=12 sin2θcos2θ,  Fxz=12 sin2θcos2θ,  Fyz=12 sin2θcos2θ,  Fzz=cos4θ.
IY=KcFiy+KaFix+KbFiz,  IZ=KcFiz+KaFix+KbFiy.

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