Abstract

We have investigated the influence of the polarization direction of excitation light on DNA sizing results obtained by a flow cytometric technique. We found strong fluorescence anisotropy of the fluorescent signal from λ DNA stained with the bis-intercalating dye TOTO-1. Small fragments of DNA are less sensitive to polarization than larger pieces. This effect is more pronounced at faster flow speeds. These observations show a noticeable orientation of the DNA molecules introduced by the shear forces in the flow system. The data are consistent with an angle between the transition moment of fluorescence of TOTO-1, and the long axis of DNA is approximately 62°.

© 1999 Optical Society of America

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  1. P. M. Goodwin, M. E. Johnson, J. C. Martin, P. W. Ambrose, B. L. Marrone, J. H. Jett, R. A. Keller, “Rapid sizing of individual fluorescently stained DNA fragments by flow cytometry,” Nucl. Acids Res. 21, 803–806 (1993).
    [CrossRef] [PubMed]
  2. A. Castro, F. R. Faifield, E. B. Shera, “Fluorescence detection and size measurement of single DNA molecules,” Anal. Chem. 65, 849–852 (1993).
    [CrossRef]
  3. J. T. Petty, M. E. Johnson, P. M. Goodwin, J. H. Jett, R. A. Keller, “Characterization of DNA size determination of small fragments by flow cytometry,” Anal. Chem. 67, 1756–1761 (1995).
    [CrossRef]
  4. A. Castro, E. B. Shera, “Single-molecule detection: applications to ultrasensitive biochemical analysis,” Appl. Opt. 34, 3218–3222 (1995).
    [CrossRef] [PubMed]
  5. 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,” Nucl. Acids Res. 24, 4202–4209 (1996).
    [CrossRef] [PubMed]
  6. H. S. Rye, S. Yue, D. E. Wemmer, M. A. Quesada, R. P. Haugland, R. A. Mathies, A. N. Glazer, “Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications,” Nucl. Acids Res. 20, 2803–2812 (1992).
    [CrossRef] [PubMed]
  7. 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]
  8. C. Carlsson, A. Larsson, M. Jonsson, B. Albinsson, B. Norden, “Optical and photophysical properties of the oxazole yellow DNA probes YO and YOYO,” J. Phys. Chem. 98, 10313–10321 (1994).
    [CrossRef]
  9. V. Kachel, H. Fellner-Feldegg, E. Menke, “Hydrodynamic properties of flow cytometry instruments,” in Flow Cytometry and Sorting, 2nd ed., M. R. Melamed, T. Lindmo, M. L. Mendelsohn, eds. (Wiley, New York, 1990), pp. 27–44.
  10. A. Agronskaia, A. Florians, K. O. van der Werf, J. M. Schins, B. G. de Grooth, J. Greve, “A photon counting device compatible with conventional flow cytometric data acquisition electronics,” Cytometry 32, 255–259 (1998).
    [CrossRef] [PubMed]
  11. T. C. Bakker Schut, A. Florians, K. O. van der Werf, B. G. de Grooth, “Flow cytometry signal processing and data acquisition with a personal computer using an RTI-800 multifunction A/D I/O board,” Rev. Sci. Instrum. 64, 3116–3120 (1993).
    [CrossRef]
  12. B. Norden, M. Kubista, T. Kurucsev, “Linear dichroism spectroscopy of nucleic acids,” Q. Rev. Biophys. 25, 51–170 (1992).
    [CrossRef] [PubMed]

1998

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

1996

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,” Nucl. Acids Res. 24, 4202–4209 (1996).
[CrossRef] [PubMed]

1995

J. T. Petty, M. E. Johnson, P. M. Goodwin, J. H. Jett, R. A. Keller, “Characterization of DNA size determination of small fragments by flow cytometry,” Anal. Chem. 67, 1756–1761 (1995).
[CrossRef]

A. Castro, E. B. Shera, “Single-molecule detection: applications to ultrasensitive biochemical analysis,” Appl. Opt. 34, 3218–3222 (1995).
[CrossRef] [PubMed]

1994

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]

C. Carlsson, A. Larsson, M. Jonsson, B. Albinsson, B. Norden, “Optical and photophysical properties of the oxazole yellow DNA probes YO and YOYO,” J. Phys. Chem. 98, 10313–10321 (1994).
[CrossRef]

1993

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

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

T. C. Bakker Schut, A. Florians, K. O. van der Werf, B. G. de Grooth, “Flow cytometry signal processing and data acquisition with a personal computer using an RTI-800 multifunction A/D I/O board,” Rev. Sci. Instrum. 64, 3116–3120 (1993).
[CrossRef]

1992

B. Norden, M. Kubista, T. Kurucsev, “Linear dichroism spectroscopy of nucleic acids,” Q. Rev. Biophys. 25, 51–170 (1992).
[CrossRef] [PubMed]

H. S. Rye, S. Yue, D. E. Wemmer, M. A. Quesada, R. P. Haugland, R. A. Mathies, A. N. Glazer, “Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications,” Nucl. Acids Res. 20, 2803–2812 (1992).
[CrossRef] [PubMed]

Agronskaia, A.

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

Albinsson, B.

C. Carlsson, A. Larsson, M. Jonsson, B. Albinsson, B. Norden, “Optical and photophysical properties of the oxazole yellow DNA probes YO and YOYO,” J. Phys. Chem. 98, 10313–10321 (1994).
[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]

Ambrose, P. W.

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

Bakker Schut, T. C.

T. C. Bakker Schut, A. Florians, K. O. van der Werf, B. G. de Grooth, “Flow cytometry signal processing and data acquisition with a personal computer using an RTI-800 multifunction A/D I/O board,” Rev. Sci. Instrum. 64, 3116–3120 (1993).
[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,” Nucl. Acids Res. 24, 4202–4209 (1996).
[CrossRef] [PubMed]

Carlsson, C.

C. Carlsson, A. Larsson, M. Jonsson, B. Albinsson, B. Norden, “Optical and photophysical properties of the oxazole yellow DNA probes YO and YOYO,” J. Phys. Chem. 98, 10313–10321 (1994).
[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 detection: applications to ultrasensitive biochemical analysis,” Appl. Opt. 34, 3218–3222 (1995).
[CrossRef] [PubMed]

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

de Grooth, B. G.

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

T. C. Bakker Schut, A. Florians, K. O. van der Werf, B. G. de Grooth, “Flow cytometry signal processing and data acquisition with a personal computer using an RTI-800 multifunction A/D I/O board,” Rev. Sci. Instrum. 64, 3116–3120 (1993).
[CrossRef]

Faifield, F. R.

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

Fellner-Feldegg, H.

V. Kachel, H. Fellner-Feldegg, E. Menke, “Hydrodynamic properties of flow cytometry instruments,” in Flow Cytometry and Sorting, 2nd ed., M. R. Melamed, T. Lindmo, M. L. Mendelsohn, eds. (Wiley, New York, 1990), pp. 27–44.

Florians, A.

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

T. C. Bakker Schut, A. Florians, K. O. van der Werf, B. G. de Grooth, “Flow cytometry signal processing and data acquisition with a personal computer using an RTI-800 multifunction A/D I/O board,” Rev. Sci. Instrum. 64, 3116–3120 (1993).
[CrossRef]

Glazer, A. N.

H. S. Rye, S. Yue, D. E. Wemmer, M. A. Quesada, R. P. Haugland, R. A. Mathies, A. N. Glazer, “Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications,” Nucl. Acids Res. 20, 2803–2812 (1992).
[CrossRef] [PubMed]

Goodwin, P. M.

J. T. Petty, M. E. Johnson, P. M. Goodwin, J. H. Jett, R. A. Keller, “Characterization of DNA size determination of small fragments by flow cytometry,” Anal. Chem. 67, 1756–1761 (1995).
[CrossRef]

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

Greve, J.

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

Haugland, R. P.

H. S. Rye, S. Yue, D. E. Wemmer, M. A. Quesada, R. P. Haugland, R. A. Mathies, A. N. Glazer, “Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications,” Nucl. Acids Res. 20, 2803–2812 (1992).
[CrossRef] [PubMed]

Huang, Z.

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,” Nucl. Acids Res. 24, 4202–4209 (1996).
[CrossRef] [PubMed]

Jett, J. H.

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,” Nucl. Acids Res. 24, 4202–4209 (1996).
[CrossRef] [PubMed]

J. T. Petty, M. E. Johnson, P. M. Goodwin, J. H. Jett, R. A. Keller, “Characterization of DNA size determination of small fragments by flow cytometry,” Anal. Chem. 67, 1756–1761 (1995).
[CrossRef]

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

Johnson, M. E.

J. T. Petty, M. E. Johnson, P. M. Goodwin, J. H. Jett, R. A. Keller, “Characterization of DNA size determination of small fragments by flow cytometry,” Anal. Chem. 67, 1756–1761 (1995).
[CrossRef]

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

Jonsson, M.

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]

C. Carlsson, A. Larsson, M. Jonsson, B. Albinsson, B. Norden, “Optical and photophysical properties of the oxazole yellow DNA probes YO and YOYO,” J. Phys. Chem. 98, 10313–10321 (1994).
[CrossRef]

Kachel, V.

V. Kachel, H. Fellner-Feldegg, E. Menke, “Hydrodynamic properties of flow cytometry instruments,” in Flow Cytometry and Sorting, 2nd ed., M. R. Melamed, T. Lindmo, M. L. Mendelsohn, eds. (Wiley, New York, 1990), pp. 27–44.

Keller, R. A.

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,” Nucl. Acids Res. 24, 4202–4209 (1996).
[CrossRef] [PubMed]

J. T. Petty, M. E. Johnson, P. M. Goodwin, J. H. Jett, R. A. Keller, “Characterization of DNA size determination of small fragments by flow cytometry,” Anal. Chem. 67, 1756–1761 (1995).
[CrossRef]

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

Kubista, M.

B. Norden, M. Kubista, T. Kurucsev, “Linear dichroism spectroscopy of nucleic acids,” Q. Rev. Biophys. 25, 51–170 (1992).
[CrossRef] [PubMed]

Kurucsev, T.

B. Norden, M. Kubista, T. Kurucsev, “Linear dichroism spectroscopy of nucleic acids,” Q. Rev. Biophys. 25, 51–170 (1992).
[CrossRef] [PubMed]

Larsson, A.

C. Carlsson, A. Larsson, M. Jonsson, B. Albinsson, B. Norden, “Optical and photophysical properties of the oxazole yellow DNA probes YO and YOYO,” J. Phys. Chem. 98, 10313–10321 (1994).
[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,” Nucl. Acids Res. 24, 4202–4209 (1996).
[CrossRef] [PubMed]

Marrone, B. L.

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

Martin, J. C.

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

Mathies, R. A.

H. S. Rye, S. Yue, D. E. Wemmer, M. A. Quesada, R. P. Haugland, R. A. Mathies, A. N. Glazer, “Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications,” Nucl. Acids Res. 20, 2803–2812 (1992).
[CrossRef] [PubMed]

Menke, E.

V. Kachel, H. Fellner-Feldegg, E. Menke, “Hydrodynamic properties of flow cytometry instruments,” in Flow Cytometry and Sorting, 2nd ed., M. R. Melamed, T. Lindmo, M. L. Mendelsohn, eds. (Wiley, New York, 1990), pp. 27–44.

Norden, B.

C. Carlsson, A. Larsson, M. Jonsson, B. Albinsson, B. Norden, “Optical and photophysical properties of the oxazole yellow DNA probes YO and YOYO,” J. Phys. Chem. 98, 10313–10321 (1994).
[CrossRef]

B. Norden, M. Kubista, T. Kurucsev, “Linear dichroism spectroscopy of nucleic acids,” Q. Rev. Biophys. 25, 51–170 (1992).
[CrossRef] [PubMed]

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,” Nucl. Acids Res. 24, 4202–4209 (1996).
[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,” Nucl. Acids Res. 24, 4202–4209 (1996).
[CrossRef] [PubMed]

J. T. Petty, M. E. Johnson, P. M. Goodwin, J. H. Jett, R. A. Keller, “Characterization of DNA size determination of small fragments by flow cytometry,” Anal. Chem. 67, 1756–1761 (1995).
[CrossRef]

Quesada, M. A.

H. S. Rye, S. Yue, D. E. Wemmer, M. A. Quesada, R. P. Haugland, R. A. Mathies, A. N. Glazer, “Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications,” Nucl. Acids Res. 20, 2803–2812 (1992).
[CrossRef] [PubMed]

Rye, H. S.

H. S. Rye, S. Yue, D. E. Wemmer, M. A. Quesada, R. P. Haugland, R. A. Mathies, A. N. Glazer, “Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications,” Nucl. Acids Res. 20, 2803–2812 (1992).
[CrossRef] [PubMed]

Schins, J. M.

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

Shera, E. B.

A. Castro, E. B. Shera, “Single-molecule detection: applications to ultrasensitive biochemical analysis,” Appl. Opt. 34, 3218–3222 (1995).
[CrossRef] [PubMed]

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

van der Werf, K. O.

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

T. C. Bakker Schut, A. Florians, K. O. van der Werf, B. G. de Grooth, “Flow cytometry signal processing and data acquisition with a personal computer using an RTI-800 multifunction A/D I/O board,” Rev. Sci. Instrum. 64, 3116–3120 (1993).
[CrossRef]

Wemmer, D. E.

H. S. Rye, S. Yue, D. E. Wemmer, M. A. Quesada, R. P. Haugland, R. A. Mathies, A. N. Glazer, “Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications,” Nucl. Acids Res. 20, 2803–2812 (1992).
[CrossRef] [PubMed]

Yue, S.

H. S. Rye, S. Yue, D. E. Wemmer, M. A. Quesada, R. P. Haugland, R. A. Mathies, A. N. Glazer, “Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications,” Nucl. Acids Res. 20, 2803–2812 (1992).
[CrossRef] [PubMed]

Anal. Chem.

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

J. T. Petty, M. E. Johnson, P. M. Goodwin, J. H. Jett, R. A. Keller, “Characterization of DNA size determination of small fragments by flow cytometry,” Anal. Chem. 67, 1756–1761 (1995).
[CrossRef]

Appl. Opt.

Cytometry

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

J. Am. Chem. Soc.

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.

C. Carlsson, A. Larsson, M. Jonsson, B. Albinsson, B. Norden, “Optical and photophysical properties of the oxazole yellow DNA probes YO and YOYO,” J. Phys. Chem. 98, 10313–10321 (1994).
[CrossRef]

Nucl. Acids Res.

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,” Nucl. Acids Res. 24, 4202–4209 (1996).
[CrossRef] [PubMed]

H. S. Rye, S. Yue, D. E. Wemmer, M. A. Quesada, R. P. Haugland, R. A. Mathies, A. N. Glazer, “Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications,” Nucl. Acids Res. 20, 2803–2812 (1992).
[CrossRef] [PubMed]

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

Q. Rev. Biophys.

B. Norden, M. Kubista, T. Kurucsev, “Linear dichroism spectroscopy of nucleic acids,” Q. Rev. Biophys. 25, 51–170 (1992).
[CrossRef] [PubMed]

Rev. Sci. Instrum.

T. C. Bakker Schut, A. Florians, K. O. van der Werf, B. G. de Grooth, “Flow cytometry signal processing and data acquisition with a personal computer using an RTI-800 multifunction A/D I/O board,” Rev. Sci. Instrum. 64, 3116–3120 (1993).
[CrossRef]

Other

V. Kachel, H. Fellner-Feldegg, E. Menke, “Hydrodynamic properties of flow cytometry instruments,” in Flow Cytometry and Sorting, 2nd ed., M. R. Melamed, T. Lindmo, M. L. Mendelsohn, eds. (Wiley, New York, 1990), pp. 27–44.

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Figures (7)

Fig. 1
Fig. 1

Design of the flow cell.

Fig. 2
Fig. 2

(a) Histograms of λ DNA stained with TOTO-1 for two light polarizations, perpendicular to the flow direction (solid curve) and parallel to the flow direction (dotted curve): laser intensity, 0.5 mW; sample flow speed, 8 mm/s; bin width, 33 counts/bin. (b) Histograms of 0.3-µm yellow fluorescent beads for two light polarizations: laser intensity, 4 mW; sample flow speed, 8 mm/s; bin width, 33 counts/bin.

Fig. 3
Fig. 3

Histograms of HindIII digest of λ DNA stained with TOTO-1. The fragment lengths in base pairs are indicated above the peaks: Laser intensity, 1.5 mW; sample flow speed, 8 mm/s. (a) Histogram obtained with perpendicular to flow direction polarized laser light. (b) Histogram obtained with parallel to flow direction polarized laser light: bin width, 20 counts/bin.

Fig. 4
Fig. 4

Plot of polarization ratio R for fragments of HindIII versus fragment length averaged over 11 measurements: sample-flow speed, 8 mm/s.

Fig. 5
Fig. 5

Polarization ratio of HindIII fragments versus fragment length for three different speeds of flow: 14, 8, and 5 mm/s. Error bars represent accuracy of determination of polarization ratios.

Fig. 6
Fig. 6

(a) Histogram of HindIII-digested λ DNA obtained with excitation polarization at 58.5°: bin width, 20 counts/bin; sample flow speed, 8 mm/s; 1/e 2 ratios of the laser beam, 8 µm; laser intensity, 0.5 mW. (b) Plot of mean burst size obtained from (a) versus fragment length. The correlation coefficient of the linear fit is 0.99985.

Fig. 7
Fig. 7

Calculated and measured ratios between different polarizations of detected fluorescence F xy (n, normal to the flow; p, parallel). (The first index is the direction of the excitation polarization; the second index is the direction of the polarization of the detected fluorescence.)

Tables (2)

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Table 1 Normalization to Highest-Intensity Values of Different Polarization Components of the Fluorescent Signal for Different Polarizations of Excitation Light

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Table 2 Parameters Obtained from the Histogram of HindIII-Digested λ DNA Measured with Excitation Polarization at 58.5° [Fig. 6(a)]

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