Abstract

A hydrofocusing head with an optical cuvette has been developed for the flow cytometer to generate complete scatter patterns of single particles at scattering angles ranging from 10° to 120°. The scatter signal has been measured as a function of the angle (a flying indicatrix) by the use of particle motion within a scanning system of the flow cytometer by the use of a single photomultiplier. Scattering data measured with the flow cytometer have been compared with those calculated from Mie theory for latex particles. A calculation algorithm has been used to estimate the size and the refractive index of spherical particles from the scattering data measured.

© 1995 Optical Society of America

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References

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  1. G. C. Salzman, S. B. Singham, R. G. Johnston, C. F. Bohren, “Light scattering and cytometry,” in Flow Cytometry and Sorting, 2nd ed., M. R. Melamed, T. Lindmo, M. L. Mendelsohn, eds. (Wiley, New York, 1990), pp. 81–107.
  2. M. Bartholdi, G. C. Salzman, R. D. Heibert, M. Kerker, “Differential light scattering photometer for rapid analysis of single particles in flow,” Appl. Opt. 19, 1573–1584 (1980).
    [CrossRef] [PubMed]
  3. I. A. Ludlow, P. H. Kaye, “A scanning diffractometer for the rapid analysis of microparticles and biological cells,” Colloid Interface Sci. 69, 571–589 (1979).
    [CrossRef]
  4. M. R. Loken, R. G. Sweet, L. A. Herzenberg, “Cell discrimination by multiangle light scattering,” J. Histochem. Cytochem. 24, 284–291 (1976).
    [CrossRef] [PubMed]
  5. L. B. Bangs, Uniform Latex Particles (Seradyn, Indianapolis, 1987), p. 16.
  6. C. F. Bohren, D. R. Huffman, Absorption and Scattering of Light by Small Particles (Wiley, New York, 1983), Appendix A, p. 609.

1980

1979

I. A. Ludlow, P. H. Kaye, “A scanning diffractometer for the rapid analysis of microparticles and biological cells,” Colloid Interface Sci. 69, 571–589 (1979).
[CrossRef]

1976

M. R. Loken, R. G. Sweet, L. A. Herzenberg, “Cell discrimination by multiangle light scattering,” J. Histochem. Cytochem. 24, 284–291 (1976).
[CrossRef] [PubMed]

Bangs, L. B.

L. B. Bangs, Uniform Latex Particles (Seradyn, Indianapolis, 1987), p. 16.

Bartholdi, M.

Bohren, C. F.

C. F. Bohren, D. R. Huffman, Absorption and Scattering of Light by Small Particles (Wiley, New York, 1983), Appendix A, p. 609.

G. C. Salzman, S. B. Singham, R. G. Johnston, C. F. Bohren, “Light scattering and cytometry,” in Flow Cytometry and Sorting, 2nd ed., M. R. Melamed, T. Lindmo, M. L. Mendelsohn, eds. (Wiley, New York, 1990), pp. 81–107.

Heibert, R. D.

Herzenberg, L. A.

M. R. Loken, R. G. Sweet, L. A. Herzenberg, “Cell discrimination by multiangle light scattering,” J. Histochem. Cytochem. 24, 284–291 (1976).
[CrossRef] [PubMed]

Huffman, D. R.

C. F. Bohren, D. R. Huffman, Absorption and Scattering of Light by Small Particles (Wiley, New York, 1983), Appendix A, p. 609.

Johnston, R. G.

G. C. Salzman, S. B. Singham, R. G. Johnston, C. F. Bohren, “Light scattering and cytometry,” in Flow Cytometry and Sorting, 2nd ed., M. R. Melamed, T. Lindmo, M. L. Mendelsohn, eds. (Wiley, New York, 1990), pp. 81–107.

Kaye, P. H.

I. A. Ludlow, P. H. Kaye, “A scanning diffractometer for the rapid analysis of microparticles and biological cells,” Colloid Interface Sci. 69, 571–589 (1979).
[CrossRef]

Kerker, M.

Loken, M. R.

M. R. Loken, R. G. Sweet, L. A. Herzenberg, “Cell discrimination by multiangle light scattering,” J. Histochem. Cytochem. 24, 284–291 (1976).
[CrossRef] [PubMed]

Ludlow, I. A.

I. A. Ludlow, P. H. Kaye, “A scanning diffractometer for the rapid analysis of microparticles and biological cells,” Colloid Interface Sci. 69, 571–589 (1979).
[CrossRef]

Salzman, G. C.

M. Bartholdi, G. C. Salzman, R. D. Heibert, M. Kerker, “Differential light scattering photometer for rapid analysis of single particles in flow,” Appl. Opt. 19, 1573–1584 (1980).
[CrossRef] [PubMed]

G. C. Salzman, S. B. Singham, R. G. Johnston, C. F. Bohren, “Light scattering and cytometry,” in Flow Cytometry and Sorting, 2nd ed., M. R. Melamed, T. Lindmo, M. L. Mendelsohn, eds. (Wiley, New York, 1990), pp. 81–107.

Singham, S. B.

G. C. Salzman, S. B. Singham, R. G. Johnston, C. F. Bohren, “Light scattering and cytometry,” in Flow Cytometry and Sorting, 2nd ed., M. R. Melamed, T. Lindmo, M. L. Mendelsohn, eds. (Wiley, New York, 1990), pp. 81–107.

Sweet, R. G.

M. R. Loken, R. G. Sweet, L. A. Herzenberg, “Cell discrimination by multiangle light scattering,” J. Histochem. Cytochem. 24, 284–291 (1976).
[CrossRef] [PubMed]

Appl. Opt.

Colloid Interface Sci.

I. A. Ludlow, P. H. Kaye, “A scanning diffractometer for the rapid analysis of microparticles and biological cells,” Colloid Interface Sci. 69, 571–589 (1979).
[CrossRef]

J. Histochem. Cytochem.

M. R. Loken, R. G. Sweet, L. A. Herzenberg, “Cell discrimination by multiangle light scattering,” J. Histochem. Cytochem. 24, 284–291 (1976).
[CrossRef] [PubMed]

Other

L. B. Bangs, Uniform Latex Particles (Seradyn, Indianapolis, 1987), p. 16.

C. F. Bohren, D. R. Huffman, Absorption and Scattering of Light by Small Particles (Wiley, New York, 1983), Appendix A, p. 609.

G. C. Salzman, S. B. Singham, R. G. Johnston, C. F. Bohren, “Light scattering and cytometry,” in Flow Cytometry and Sorting, 2nd ed., M. R. Melamed, T. Lindmo, M. L. Mendelsohn, eds. (Wiley, New York, 1990), pp. 81–107.

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Figures (5)

Fig. 1
Fig. 1

Schematic diagram of the hydrofocusing head with optical cuvette.

Fig. 2
Fig. 2

Simplified diagram of the scanning scatter system. The main and the trigger laser beams and the scattered rays are shown as dashed lines. After passing points 0, 1, and 2, the particle activates the detecting system and scatters light at angles of θ1 and θ2.

Fig. 3
Fig. 3

Transfer coefficient (solid curve) and the scattering angle (dashed curve) as a function of particle location (distance from the spherical reflector bottom).

Fig. 4
Fig. 4

Flying light-scattering indicatrix (squares) of a single latex particle as a) observed and b) transformed. The theoretical bestfit indicatrix (solid curve) that corresponds to sphere diameter 3 μm and refractive index 1.58.

Fig. 5
Fig. 5

Flying light-scattering indicatrix (squares) of a single latex particle as a) observed and b) transformed. The theoretical bestfit indicatrix (solid curve) that corresponds to sphere diameter 4.68 μm and refractive index 1.58.

Equations (2)

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d ( μ m ) = α 1 + α 2 [ Δ θ 2 ( 20 ) ] 1 + α 3 [ Δ θ 2 ( 20 ) ] 5 + α 4 [ Δ θ 2 ( 20 ) ] 6 ,
n = β 1 + β 2 [ V ( 30 ) ] 1 / 2 + β 3 V ( 30 ) + β 4 [ V ( 30 ) ] 2 ,

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