Abstract

A differential light scattering photometer has been developed for rapid size analysis of single particles in flow. A fluid stream carrying individual particles in single file intersects a focused laser beam at the primary focal point of an annular strip of an ellipsoidal reflector situated in a scattering chamber. The light scattered from polar angles θ = 2.5–177.5° at azimuthal angles ϕ = 0 and 180°, spanning a circle of 355°, is reflected onto a circular array of 60 photodiodes. The signal processing electronics and computer storage can accept 32 signals/particle at rates up to 1000 particles/sec. Photometer performance is tested by comparing measured responses from individual spherical particles with angular scattering patterns calculated for the particular detector geometry. These patterns exhibit the required symmetry in the two half scattering planes. Response measurements for eight samples with particle diameters of 1.1, 2.7, 5.0, 7.9, 10.0, 12.5, 15.6, and 19.5 μm are consistent with calculated size-response curves. The composition of a mixture of five components with particle diameters of 1.1, 5.0, 10.0, 15.6, and 19.5 μm is determined from an analysis of light scattering measurements at various forward-scattering angles.

© 1980 Optical Society of America

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  1. Proceedings, Sixth Engineering Foundation Conference on Automated Cytology, J. Histochem. Cytochem.27 (1979), contains 74 papers dedicated to flow cytometric methods.
  2. T. M. Jovin, S. J. Morris, G. Striker, H. A. Schultens, M. Digweed, D. J. Arndt-Jovin, J. Histochem. Cytochem. 24, 269 (1976).
    [CrossRef] [PubMed]
  3. M. Kerker, The Scattering of Light and Other Electromagnetic Radiation (Academic, New York, 1969).
  4. M. Kerker, H. Chew, P. J. McNulty, J. P. Kratohvil, D. D. Cooke, M. Sculley, M.-P. Lee, J. Histochem. Cytochem. 27, 250 (1979);M. Kerker, S. D. Druger, Appl. Opt. 18, 1172 (1979).
    [CrossRef] [PubMed]
  5. W. S. Bickel, J. F. Davidson, D. R. Huffman, R. Kilkson, Proc. Natl. Acad. Sci. USA 73, 486 (1976).
    [CrossRef] [PubMed]
  6. M. Kerker, D. D. Cooke, H. Chew, P. J. McNulty, J. Opt. Soc. Am. 68, 592 (1978).
    [CrossRef]
  7. G. C. Salzman, J. M. Crowell, C. A. Goad, K. M. Hansen, R. D. Hiebert, P. M. LaBauve, J. C. Martin, M. L. Ingram, P. F. Mullaney, Clin. Chem. 21, 1297 (1975).
    [PubMed]
  8. F. T. Gucker, J. Tuma, H.-M. Lin, C.-M. Huang, S. C. Ems, T. R. Marshall, J. Aerosol Sci. 4, 389 (1973).
    [CrossRef]
  9. M. J. Skogen-Hagenson, G. C. Salzman, P. F. Mullaney, W. H. Brockman, J. Histochem. Cytochem. 25, 784 (1977).
    [CrossRef] [PubMed]
  10. P. J. Crossland-Taylor, Nature 171, 37 (1953).
    [CrossRef]
  11. J. P. Kratohvil, J. Colloid Interface Sci. 21, 498 (1966).
    [CrossRef]
  12. J. M. Crowell, R. D. Hiebert, G. C. Salzman, B. J. Price, L. S. Cram, P. F. Mullaney, IEEE Trans. Biomed. Eng. BME-25, 519 (1978).
    [CrossRef]
  13. T. P. Wallace, J. P. Kratohvil, J. Polymer Sci. 8A-2, 1425 (1970).

1979

M. Kerker, H. Chew, P. J. McNulty, J. P. Kratohvil, D. D. Cooke, M. Sculley, M.-P. Lee, J. Histochem. Cytochem. 27, 250 (1979);M. Kerker, S. D. Druger, Appl. Opt. 18, 1172 (1979).
[CrossRef] [PubMed]

1978

J. M. Crowell, R. D. Hiebert, G. C. Salzman, B. J. Price, L. S. Cram, P. F. Mullaney, IEEE Trans. Biomed. Eng. BME-25, 519 (1978).
[CrossRef]

M. Kerker, D. D. Cooke, H. Chew, P. J. McNulty, J. Opt. Soc. Am. 68, 592 (1978).
[CrossRef]

1977

M. J. Skogen-Hagenson, G. C. Salzman, P. F. Mullaney, W. H. Brockman, J. Histochem. Cytochem. 25, 784 (1977).
[CrossRef] [PubMed]

1976

T. M. Jovin, S. J. Morris, G. Striker, H. A. Schultens, M. Digweed, D. J. Arndt-Jovin, J. Histochem. Cytochem. 24, 269 (1976).
[CrossRef] [PubMed]

W. S. Bickel, J. F. Davidson, D. R. Huffman, R. Kilkson, Proc. Natl. Acad. Sci. USA 73, 486 (1976).
[CrossRef] [PubMed]

1975

G. C. Salzman, J. M. Crowell, C. A. Goad, K. M. Hansen, R. D. Hiebert, P. M. LaBauve, J. C. Martin, M. L. Ingram, P. F. Mullaney, Clin. Chem. 21, 1297 (1975).
[PubMed]

1973

F. T. Gucker, J. Tuma, H.-M. Lin, C.-M. Huang, S. C. Ems, T. R. Marshall, J. Aerosol Sci. 4, 389 (1973).
[CrossRef]

1970

T. P. Wallace, J. P. Kratohvil, J. Polymer Sci. 8A-2, 1425 (1970).

1966

J. P. Kratohvil, J. Colloid Interface Sci. 21, 498 (1966).
[CrossRef]

1953

P. J. Crossland-Taylor, Nature 171, 37 (1953).
[CrossRef]

Arndt-Jovin, D. J.

T. M. Jovin, S. J. Morris, G. Striker, H. A. Schultens, M. Digweed, D. J. Arndt-Jovin, J. Histochem. Cytochem. 24, 269 (1976).
[CrossRef] [PubMed]

Bickel, W. S.

W. S. Bickel, J. F. Davidson, D. R. Huffman, R. Kilkson, Proc. Natl. Acad. Sci. USA 73, 486 (1976).
[CrossRef] [PubMed]

Brockman, W. H.

M. J. Skogen-Hagenson, G. C. Salzman, P. F. Mullaney, W. H. Brockman, J. Histochem. Cytochem. 25, 784 (1977).
[CrossRef] [PubMed]

Chew, H.

M. Kerker, H. Chew, P. J. McNulty, J. P. Kratohvil, D. D. Cooke, M. Sculley, M.-P. Lee, J. Histochem. Cytochem. 27, 250 (1979);M. Kerker, S. D. Druger, Appl. Opt. 18, 1172 (1979).
[CrossRef] [PubMed]

M. Kerker, D. D. Cooke, H. Chew, P. J. McNulty, J. Opt. Soc. Am. 68, 592 (1978).
[CrossRef]

Cooke, D. D.

M. Kerker, H. Chew, P. J. McNulty, J. P. Kratohvil, D. D. Cooke, M. Sculley, M.-P. Lee, J. Histochem. Cytochem. 27, 250 (1979);M. Kerker, S. D. Druger, Appl. Opt. 18, 1172 (1979).
[CrossRef] [PubMed]

M. Kerker, D. D. Cooke, H. Chew, P. J. McNulty, J. Opt. Soc. Am. 68, 592 (1978).
[CrossRef]

Cram, L. S.

J. M. Crowell, R. D. Hiebert, G. C. Salzman, B. J. Price, L. S. Cram, P. F. Mullaney, IEEE Trans. Biomed. Eng. BME-25, 519 (1978).
[CrossRef]

Crossland-Taylor, P. J.

P. J. Crossland-Taylor, Nature 171, 37 (1953).
[CrossRef]

Crowell, J. M.

J. M. Crowell, R. D. Hiebert, G. C. Salzman, B. J. Price, L. S. Cram, P. F. Mullaney, IEEE Trans. Biomed. Eng. BME-25, 519 (1978).
[CrossRef]

G. C. Salzman, J. M. Crowell, C. A. Goad, K. M. Hansen, R. D. Hiebert, P. M. LaBauve, J. C. Martin, M. L. Ingram, P. F. Mullaney, Clin. Chem. 21, 1297 (1975).
[PubMed]

Davidson, J. F.

W. S. Bickel, J. F. Davidson, D. R. Huffman, R. Kilkson, Proc. Natl. Acad. Sci. USA 73, 486 (1976).
[CrossRef] [PubMed]

Digweed, M.

T. M. Jovin, S. J. Morris, G. Striker, H. A. Schultens, M. Digweed, D. J. Arndt-Jovin, J. Histochem. Cytochem. 24, 269 (1976).
[CrossRef] [PubMed]

Ems, S. C.

F. T. Gucker, J. Tuma, H.-M. Lin, C.-M. Huang, S. C. Ems, T. R. Marshall, J. Aerosol Sci. 4, 389 (1973).
[CrossRef]

Goad, C. A.

G. C. Salzman, J. M. Crowell, C. A. Goad, K. M. Hansen, R. D. Hiebert, P. M. LaBauve, J. C. Martin, M. L. Ingram, P. F. Mullaney, Clin. Chem. 21, 1297 (1975).
[PubMed]

Gucker, F. T.

F. T. Gucker, J. Tuma, H.-M. Lin, C.-M. Huang, S. C. Ems, T. R. Marshall, J. Aerosol Sci. 4, 389 (1973).
[CrossRef]

Hansen, K. M.

G. C. Salzman, J. M. Crowell, C. A. Goad, K. M. Hansen, R. D. Hiebert, P. M. LaBauve, J. C. Martin, M. L. Ingram, P. F. Mullaney, Clin. Chem. 21, 1297 (1975).
[PubMed]

Hiebert, R. D.

J. M. Crowell, R. D. Hiebert, G. C. Salzman, B. J. Price, L. S. Cram, P. F. Mullaney, IEEE Trans. Biomed. Eng. BME-25, 519 (1978).
[CrossRef]

G. C. Salzman, J. M. Crowell, C. A. Goad, K. M. Hansen, R. D. Hiebert, P. M. LaBauve, J. C. Martin, M. L. Ingram, P. F. Mullaney, Clin. Chem. 21, 1297 (1975).
[PubMed]

Huang, C.-M.

F. T. Gucker, J. Tuma, H.-M. Lin, C.-M. Huang, S. C. Ems, T. R. Marshall, J. Aerosol Sci. 4, 389 (1973).
[CrossRef]

Huffman, D. R.

W. S. Bickel, J. F. Davidson, D. R. Huffman, R. Kilkson, Proc. Natl. Acad. Sci. USA 73, 486 (1976).
[CrossRef] [PubMed]

Ingram, M. L.

G. C. Salzman, J. M. Crowell, C. A. Goad, K. M. Hansen, R. D. Hiebert, P. M. LaBauve, J. C. Martin, M. L. Ingram, P. F. Mullaney, Clin. Chem. 21, 1297 (1975).
[PubMed]

Jovin, T. M.

T. M. Jovin, S. J. Morris, G. Striker, H. A. Schultens, M. Digweed, D. J. Arndt-Jovin, J. Histochem. Cytochem. 24, 269 (1976).
[CrossRef] [PubMed]

Kerker, M.

M. Kerker, H. Chew, P. J. McNulty, J. P. Kratohvil, D. D. Cooke, M. Sculley, M.-P. Lee, J. Histochem. Cytochem. 27, 250 (1979);M. Kerker, S. D. Druger, Appl. Opt. 18, 1172 (1979).
[CrossRef] [PubMed]

M. Kerker, D. D. Cooke, H. Chew, P. J. McNulty, J. Opt. Soc. Am. 68, 592 (1978).
[CrossRef]

M. Kerker, The Scattering of Light and Other Electromagnetic Radiation (Academic, New York, 1969).

Kilkson, R.

W. S. Bickel, J. F. Davidson, D. R. Huffman, R. Kilkson, Proc. Natl. Acad. Sci. USA 73, 486 (1976).
[CrossRef] [PubMed]

Kratohvil, J. P.

M. Kerker, H. Chew, P. J. McNulty, J. P. Kratohvil, D. D. Cooke, M. Sculley, M.-P. Lee, J. Histochem. Cytochem. 27, 250 (1979);M. Kerker, S. D. Druger, Appl. Opt. 18, 1172 (1979).
[CrossRef] [PubMed]

T. P. Wallace, J. P. Kratohvil, J. Polymer Sci. 8A-2, 1425 (1970).

J. P. Kratohvil, J. Colloid Interface Sci. 21, 498 (1966).
[CrossRef]

LaBauve, P. M.

G. C. Salzman, J. M. Crowell, C. A. Goad, K. M. Hansen, R. D. Hiebert, P. M. LaBauve, J. C. Martin, M. L. Ingram, P. F. Mullaney, Clin. Chem. 21, 1297 (1975).
[PubMed]

Lee, M.-P.

M. Kerker, H. Chew, P. J. McNulty, J. P. Kratohvil, D. D. Cooke, M. Sculley, M.-P. Lee, J. Histochem. Cytochem. 27, 250 (1979);M. Kerker, S. D. Druger, Appl. Opt. 18, 1172 (1979).
[CrossRef] [PubMed]

Lin, H.-M.

F. T. Gucker, J. Tuma, H.-M. Lin, C.-M. Huang, S. C. Ems, T. R. Marshall, J. Aerosol Sci. 4, 389 (1973).
[CrossRef]

Marshall, T. R.

F. T. Gucker, J. Tuma, H.-M. Lin, C.-M. Huang, S. C. Ems, T. R. Marshall, J. Aerosol Sci. 4, 389 (1973).
[CrossRef]

Martin, J. C.

G. C. Salzman, J. M. Crowell, C. A. Goad, K. M. Hansen, R. D. Hiebert, P. M. LaBauve, J. C. Martin, M. L. Ingram, P. F. Mullaney, Clin. Chem. 21, 1297 (1975).
[PubMed]

McNulty, P. J.

M. Kerker, H. Chew, P. J. McNulty, J. P. Kratohvil, D. D. Cooke, M. Sculley, M.-P. Lee, J. Histochem. Cytochem. 27, 250 (1979);M. Kerker, S. D. Druger, Appl. Opt. 18, 1172 (1979).
[CrossRef] [PubMed]

M. Kerker, D. D. Cooke, H. Chew, P. J. McNulty, J. Opt. Soc. Am. 68, 592 (1978).
[CrossRef]

Morris, S. J.

T. M. Jovin, S. J. Morris, G. Striker, H. A. Schultens, M. Digweed, D. J. Arndt-Jovin, J. Histochem. Cytochem. 24, 269 (1976).
[CrossRef] [PubMed]

Mullaney, P. F.

J. M. Crowell, R. D. Hiebert, G. C. Salzman, B. J. Price, L. S. Cram, P. F. Mullaney, IEEE Trans. Biomed. Eng. BME-25, 519 (1978).
[CrossRef]

M. J. Skogen-Hagenson, G. C. Salzman, P. F. Mullaney, W. H. Brockman, J. Histochem. Cytochem. 25, 784 (1977).
[CrossRef] [PubMed]

G. C. Salzman, J. M. Crowell, C. A. Goad, K. M. Hansen, R. D. Hiebert, P. M. LaBauve, J. C. Martin, M. L. Ingram, P. F. Mullaney, Clin. Chem. 21, 1297 (1975).
[PubMed]

Price, B. J.

J. M. Crowell, R. D. Hiebert, G. C. Salzman, B. J. Price, L. S. Cram, P. F. Mullaney, IEEE Trans. Biomed. Eng. BME-25, 519 (1978).
[CrossRef]

Salzman, G. C.

J. M. Crowell, R. D. Hiebert, G. C. Salzman, B. J. Price, L. S. Cram, P. F. Mullaney, IEEE Trans. Biomed. Eng. BME-25, 519 (1978).
[CrossRef]

M. J. Skogen-Hagenson, G. C. Salzman, P. F. Mullaney, W. H. Brockman, J. Histochem. Cytochem. 25, 784 (1977).
[CrossRef] [PubMed]

G. C. Salzman, J. M. Crowell, C. A. Goad, K. M. Hansen, R. D. Hiebert, P. M. LaBauve, J. C. Martin, M. L. Ingram, P. F. Mullaney, Clin. Chem. 21, 1297 (1975).
[PubMed]

Schultens, H. A.

T. M. Jovin, S. J. Morris, G. Striker, H. A. Schultens, M. Digweed, D. J. Arndt-Jovin, J. Histochem. Cytochem. 24, 269 (1976).
[CrossRef] [PubMed]

Sculley, M.

M. Kerker, H. Chew, P. J. McNulty, J. P. Kratohvil, D. D. Cooke, M. Sculley, M.-P. Lee, J. Histochem. Cytochem. 27, 250 (1979);M. Kerker, S. D. Druger, Appl. Opt. 18, 1172 (1979).
[CrossRef] [PubMed]

Skogen-Hagenson, M. J.

M. J. Skogen-Hagenson, G. C. Salzman, P. F. Mullaney, W. H. Brockman, J. Histochem. Cytochem. 25, 784 (1977).
[CrossRef] [PubMed]

Striker, G.

T. M. Jovin, S. J. Morris, G. Striker, H. A. Schultens, M. Digweed, D. J. Arndt-Jovin, J. Histochem. Cytochem. 24, 269 (1976).
[CrossRef] [PubMed]

Tuma, J.

F. T. Gucker, J. Tuma, H.-M. Lin, C.-M. Huang, S. C. Ems, T. R. Marshall, J. Aerosol Sci. 4, 389 (1973).
[CrossRef]

Wallace, T. P.

T. P. Wallace, J. P. Kratohvil, J. Polymer Sci. 8A-2, 1425 (1970).

Clin. Chem.

G. C. Salzman, J. M. Crowell, C. A. Goad, K. M. Hansen, R. D. Hiebert, P. M. LaBauve, J. C. Martin, M. L. Ingram, P. F. Mullaney, Clin. Chem. 21, 1297 (1975).
[PubMed]

IEEE Trans. Biomed. Eng.

J. M. Crowell, R. D. Hiebert, G. C. Salzman, B. J. Price, L. S. Cram, P. F. Mullaney, IEEE Trans. Biomed. Eng. BME-25, 519 (1978).
[CrossRef]

J. Aerosol Sci.

F. T. Gucker, J. Tuma, H.-M. Lin, C.-M. Huang, S. C. Ems, T. R. Marshall, J. Aerosol Sci. 4, 389 (1973).
[CrossRef]

J. Colloid Interface Sci.

J. P. Kratohvil, J. Colloid Interface Sci. 21, 498 (1966).
[CrossRef]

J. Histochem. Cytochem.

M. J. Skogen-Hagenson, G. C. Salzman, P. F. Mullaney, W. H. Brockman, J. Histochem. Cytochem. 25, 784 (1977).
[CrossRef] [PubMed]

T. M. Jovin, S. J. Morris, G. Striker, H. A. Schultens, M. Digweed, D. J. Arndt-Jovin, J. Histochem. Cytochem. 24, 269 (1976).
[CrossRef] [PubMed]

M. Kerker, H. Chew, P. J. McNulty, J. P. Kratohvil, D. D. Cooke, M. Sculley, M.-P. Lee, J. Histochem. Cytochem. 27, 250 (1979);M. Kerker, S. D. Druger, Appl. Opt. 18, 1172 (1979).
[CrossRef] [PubMed]

J. Opt. Soc. Am.

J. Polymer Sci.

T. P. Wallace, J. P. Kratohvil, J. Polymer Sci. 8A-2, 1425 (1970).

Nature

P. J. Crossland-Taylor, Nature 171, 37 (1953).
[CrossRef]

Proc. Natl. Acad. Sci. USA

W. S. Bickel, J. F. Davidson, D. R. Huffman, R. Kilkson, Proc. Natl. Acad. Sci. USA 73, 486 (1976).
[CrossRef] [PubMed]

Other

M. Kerker, The Scattering of Light and Other Electromagnetic Radiation (Academic, New York, 1969).

Proceedings, Sixth Engineering Foundation Conference on Automated Cytology, J. Histochem. Cytochem.27 (1979), contains 74 papers dedicated to flow cytometric methods.

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Figures (15)

Fig. 1
Fig. 1

Optical basis of the instrument showing the intersection of the laser beam and particle stream at the primary focus of a circular strip of an ellipsoidal reflector and also the reflected scattered rays converging to the detector array (not to scale).

Fig. 2
Fig. 2

Cross-sectional and front views of the chamber showing the ellipsoidal reflector, flow nozzles, and Rayleigh horn.

Fig. 3
Fig. 3

Cross section of the scattering chamber showing the light path and reflections. The light scattering event occurs at F1. Path of the detected ray is illustrated. ϕ1 is the angle of incidence on the ellipsoidal reflector, ϕ2 is the angle of incidence at the saline n1–quartz window n2 interface, ϕ3 is the angle of incidence at the quartz window–air n3 interface, and ϕ4 is the angle of refraction of the emerging light. Reflections from each interface are stopped by the mask.

Fig. 4
Fig. 4

Schematic diagram of detector array showing numbering of the elements and the subtended angle.

Fig. 5
Fig. 5

Ten measured scattering patterns, each from an individual polystyrene latex particle in a narrowly dispersed population (nominal diameter 12.5 μm, nominal size parameter α = 102.1).

Fig. 6
Fig. 6

Measured angular scattering pattern (points) from a single particle in a polymethylmethacrylate latex sample (9.55-μm nominal diam) compared with a theoretical curve corresponding to instrument geometry and α = 78.0, m = 1.12.

Fig. 7
Fig. 7

Measured angular scattering pattern (points) from a single particle in a polystyrene latex sample (12.5-μm nominal diam) compared with a theoretical curve corresponding to instrument geometry and α = 102.1, m = 1.20.

Fig. 8
Fig. 8

Measured angular scattering pattern (points) from a single particle in a polystyrene latex sample (19.5-μm nominal diam) compared with a theoretical curve corresponding to instrument geometry and α = 159.4, m = 1.20.

Fig. 9
Fig. 9

Scattering pattern over the full circle of scattered signals showing the symmetry about 180°; polymer latex, nominal size 12.5 μm, nominal size parameter α = 102.1.

Fig. 10
Fig. 10

Same as Fig. 9 for nominal size 15.6 μm, nominal size parameter α = 127.5.

Fig. 11
Fig. 11

Polar plot of angular scattering function i2 vs scattering angle θ for α = 102.1, m = 1.20.

Fig. 12
Fig. 12

Response curve at detector 5 (Table I) calculated for instrument geometry. Points are relative measured signals for eight latex systems plotted against nominal size parameter.

Fig. 13
Fig. 13

Pulse height distributions at detectors 1–8 for sample 8 (1.1 μm) (Table II) showing migration to lower channel numbers with increasing scattering angle.

Fig. 14
Fig. 14

Pulse height distributions at detectors 1–8 for sample 4 (10.0 μm.) (Table II) showing saturation at detectors 1–4 and migration to lower channel numbers with increasing scattering angle.

Fig. 15
Fig. 15

Pulse height distributions at detectors 1–8 for a mixture of samples 1, 2, 4, 6, 8 (Table II).

Tables (3)

Tables Icon

Table I Angular Ranges Subtended by Detectorsa

Tables Icon

Table II Nominal Diameters of Polystyrene Latex Particles Used in this Work

Tables Icon

Table III Comparison of Ratios of Component Particle Number Determined by Light Scattering with that Obtained by Coulter Counts of the Single Components

Metrics