Abstract

A flow cytometer was used to estimate the relative real refractive indices of six species of phytoplankton cells. Light scatter measurements at 90° and a near-forward region (1.5–19°) were made with hydrocarbon dispersions and a glass bead suspension having known refractive indices. The ratios of the intensity of light scatter in these two angular regions for these standards was found (and can be shown, theoretically) to be proportional to the refractive index of the material. Using these calibration materials, light scatter measurements of individual phytoplankton cells yielded estimates of the relative refractive indices of these algae. These measurements are well within the historically accepted range of algal refractive indices, however, a significant degree of interspecific variability exists. The resolution of this technique to measure relative real refractive index (±0.005) represents a new capability in the assessment of optical properties of marine particulates.

© 1986 Optical Society of America

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References

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  1. R. W. Sheldon, T. R. Parsons, A Practical Manual on the Use of the Coulter Counter in Marine Science (Coulter Electronics Sales Co., Toronto, 1967).
  2. B. J. Trask, G. J. Van Den Engh, J. H. B. W. Elgershuizen, “Analysis of Phytoplankton by Flow Cytometry,” Cytometry 2, 258 (1982).
  3. J. H. Paul, W. H. Jeffrey, “Measurement of Diameters of Estuarine Bacteria and Particulates in Natural Water Samples by Use of a Submicron Particle Analyzer,” Curr. Microbiol. 10, 7 (1984).
  4. M. E. Sieracki, P. W. Johnson, J. M. Sieburth, “Detection, Enumeration and Sizing of Planktonic Bacteria by Image Analyzed Epifluorescence Microscopy,” Appl. Environ. Microbiol. 49, 799 (1985).
  5. K. L. Carder, R. D. Tomlinson, G. F. Beardsley, “A Technique for the Estimation of the Indices of Refraction of Marine Phytoplankters,” Limnol. Oceanogr. 17, 833 (1972).
  6. J. R. V. Zaneveld, D. M. Roach, H. Pak, “The Determination of the Index of Refraction Distribution of Oceanic Particulates,” J. Geophys. Res. 79, 4091 (1974).
  7. M. Tanaka, T. Nakajima, T. Takamura, “Simultaneous Determination of Complex Refractive Index and Size Distribution of Airborne and Water Suspended Particles from Light Scattering Measurements,” J. Meteorol. Soc. Jpn. 60, 1259 (1982).
  8. N. G. Jerlov, Marine Optics (Elsevier, Amsterdam, 1976).
  9. O. B. Brown, H. R. Gordon, “Tables of Mie scattering Functions for Low Index Particles Suspended in Water,” U. Miami Optical Physics Laboratory, Publication M1APW-OP-765 (1971).
  10. Using Brown and Gordon Tables and an assumed concentration of 104 cells/mliter of 5-μm diameter and respective relative indices of refraction of 1.01 and 1.03.
  11. C. M. Yentsch, C. S. Yentsch, “Emergence of Optical Instrumentation for Measuring Biological Properties,” Oceanogr. Mar. Biol. Ann. Rev. 22, 55 (1984).
  12. C. M. Yentsch et al., “Flow Cytometry and Sorting: A Technique for Analysis and Sorting of Aquatic Particles,” Limnol. Oceanogr. 28, 1275 (1983).
  13. G. C. Salzman, P. F. Mullaney, B. J. Brice, “Light Scattering Approaches to Cell Characterization,” in Flow Cytometry and Sorting, M. R. Melamed, P. F. Mullaney, M. L. Mendelsohn, Eds. (Wiley, New York, 1979).
  14. B. J. Price, V. H. Kollman, G. C. Salzman, “Light Scatter Analysis of Microalgae,” Biophys. J. 22, 29 (1978).
  15. D. H. Tycko, M. H. Metz, E. A. Epstein, A. Grinbaum, “Flow-Cytometric Light Scattering Measurement of Red Blood Cell Volume and Hemoglobin Concentration,” Appl. Opt. 24, 1355 (1985).
  16. E. Haugen, R. Selvin, Assistant Curator of the Center for Culture of Marine Phytoplankton; personal communication.
  17. R. R. L. Guillard, J. Ryther, “Studies on Marine Planktonic Diatoms I. Cyclotella nana Hustedt and Detonula confervacea (cleve) Gran,” Can. J. Microbiol. 8, 229 (1962).
  18. P. Latimer, “Light Scattering by a Structured Particle: the Homogeneous Sphere with Holes,” Appl. Opt. 23, 1844 (1984).
  19. P. Latimer, “Light Scattering by a Homogeneous Sphere with Radial Projections,” Appl. Opt. 23, 442 (1984).
  20. S. Asano, M. Sato, “Light Scattering by Randomly Oriented Spheroidal Particles,” Appl. Opt. 19, 962 (1980).
  21. S. Asano, “Light Scattering by Horizontally Oriented Spheroidal Particles,” Appl. Opt. 22, 1390 (1983).

1985 (2)

M. E. Sieracki, P. W. Johnson, J. M. Sieburth, “Detection, Enumeration and Sizing of Planktonic Bacteria by Image Analyzed Epifluorescence Microscopy,” Appl. Environ. Microbiol. 49, 799 (1985).

D. H. Tycko, M. H. Metz, E. A. Epstein, A. Grinbaum, “Flow-Cytometric Light Scattering Measurement of Red Blood Cell Volume and Hemoglobin Concentration,” Appl. Opt. 24, 1355 (1985).

1984 (4)

P. Latimer, “Light Scattering by a Structured Particle: the Homogeneous Sphere with Holes,” Appl. Opt. 23, 1844 (1984).

P. Latimer, “Light Scattering by a Homogeneous Sphere with Radial Projections,” Appl. Opt. 23, 442 (1984).

J. H. Paul, W. H. Jeffrey, “Measurement of Diameters of Estuarine Bacteria and Particulates in Natural Water Samples by Use of a Submicron Particle Analyzer,” Curr. Microbiol. 10, 7 (1984).

C. M. Yentsch, C. S. Yentsch, “Emergence of Optical Instrumentation for Measuring Biological Properties,” Oceanogr. Mar. Biol. Ann. Rev. 22, 55 (1984).

1983 (2)

C. M. Yentsch et al., “Flow Cytometry and Sorting: A Technique for Analysis and Sorting of Aquatic Particles,” Limnol. Oceanogr. 28, 1275 (1983).

S. Asano, “Light Scattering by Horizontally Oriented Spheroidal Particles,” Appl. Opt. 22, 1390 (1983).

1982 (2)

M. Tanaka, T. Nakajima, T. Takamura, “Simultaneous Determination of Complex Refractive Index and Size Distribution of Airborne and Water Suspended Particles from Light Scattering Measurements,” J. Meteorol. Soc. Jpn. 60, 1259 (1982).

B. J. Trask, G. J. Van Den Engh, J. H. B. W. Elgershuizen, “Analysis of Phytoplankton by Flow Cytometry,” Cytometry 2, 258 (1982).

1980 (1)

1978 (1)

B. J. Price, V. H. Kollman, G. C. Salzman, “Light Scatter Analysis of Microalgae,” Biophys. J. 22, 29 (1978).

1974 (1)

J. R. V. Zaneveld, D. M. Roach, H. Pak, “The Determination of the Index of Refraction Distribution of Oceanic Particulates,” J. Geophys. Res. 79, 4091 (1974).

1972 (1)

K. L. Carder, R. D. Tomlinson, G. F. Beardsley, “A Technique for the Estimation of the Indices of Refraction of Marine Phytoplankters,” Limnol. Oceanogr. 17, 833 (1972).

1962 (1)

R. R. L. Guillard, J. Ryther, “Studies on Marine Planktonic Diatoms I. Cyclotella nana Hustedt and Detonula confervacea (cleve) Gran,” Can. J. Microbiol. 8, 229 (1962).

Asano, S.

Beardsley, G. F.

K. L. Carder, R. D. Tomlinson, G. F. Beardsley, “A Technique for the Estimation of the Indices of Refraction of Marine Phytoplankters,” Limnol. Oceanogr. 17, 833 (1972).

Brice, B. J.

G. C. Salzman, P. F. Mullaney, B. J. Brice, “Light Scattering Approaches to Cell Characterization,” in Flow Cytometry and Sorting, M. R. Melamed, P. F. Mullaney, M. L. Mendelsohn, Eds. (Wiley, New York, 1979).

Brown, O. B.

O. B. Brown, H. R. Gordon, “Tables of Mie scattering Functions for Low Index Particles Suspended in Water,” U. Miami Optical Physics Laboratory, Publication M1APW-OP-765 (1971).

Carder, K. L.

K. L. Carder, R. D. Tomlinson, G. F. Beardsley, “A Technique for the Estimation of the Indices of Refraction of Marine Phytoplankters,” Limnol. Oceanogr. 17, 833 (1972).

Elgershuizen, J. H. B. W.

B. J. Trask, G. J. Van Den Engh, J. H. B. W. Elgershuizen, “Analysis of Phytoplankton by Flow Cytometry,” Cytometry 2, 258 (1982).

Epstein, E. A.

Gordon, H. R.

O. B. Brown, H. R. Gordon, “Tables of Mie scattering Functions for Low Index Particles Suspended in Water,” U. Miami Optical Physics Laboratory, Publication M1APW-OP-765 (1971).

Grinbaum, A.

Guillard, R. R. L.

R. R. L. Guillard, J. Ryther, “Studies on Marine Planktonic Diatoms I. Cyclotella nana Hustedt and Detonula confervacea (cleve) Gran,” Can. J. Microbiol. 8, 229 (1962).

Haugen, E.

E. Haugen, R. Selvin, Assistant Curator of the Center for Culture of Marine Phytoplankton; personal communication.

Jeffrey, W. H.

J. H. Paul, W. H. Jeffrey, “Measurement of Diameters of Estuarine Bacteria and Particulates in Natural Water Samples by Use of a Submicron Particle Analyzer,” Curr. Microbiol. 10, 7 (1984).

Jerlov, N. G.

N. G. Jerlov, Marine Optics (Elsevier, Amsterdam, 1976).

Johnson, P. W.

M. E. Sieracki, P. W. Johnson, J. M. Sieburth, “Detection, Enumeration and Sizing of Planktonic Bacteria by Image Analyzed Epifluorescence Microscopy,” Appl. Environ. Microbiol. 49, 799 (1985).

Kollman, V. H.

B. J. Price, V. H. Kollman, G. C. Salzman, “Light Scatter Analysis of Microalgae,” Biophys. J. 22, 29 (1978).

Latimer, P.

Metz, M. H.

Mullaney, P. F.

G. C. Salzman, P. F. Mullaney, B. J. Brice, “Light Scattering Approaches to Cell Characterization,” in Flow Cytometry and Sorting, M. R. Melamed, P. F. Mullaney, M. L. Mendelsohn, Eds. (Wiley, New York, 1979).

Nakajima, T.

M. Tanaka, T. Nakajima, T. Takamura, “Simultaneous Determination of Complex Refractive Index and Size Distribution of Airborne and Water Suspended Particles from Light Scattering Measurements,” J. Meteorol. Soc. Jpn. 60, 1259 (1982).

Pak, H.

J. R. V. Zaneveld, D. M. Roach, H. Pak, “The Determination of the Index of Refraction Distribution of Oceanic Particulates,” J. Geophys. Res. 79, 4091 (1974).

Parsons, T. R.

R. W. Sheldon, T. R. Parsons, A Practical Manual on the Use of the Coulter Counter in Marine Science (Coulter Electronics Sales Co., Toronto, 1967).

Paul, J. H.

J. H. Paul, W. H. Jeffrey, “Measurement of Diameters of Estuarine Bacteria and Particulates in Natural Water Samples by Use of a Submicron Particle Analyzer,” Curr. Microbiol. 10, 7 (1984).

Price, B. J.

B. J. Price, V. H. Kollman, G. C. Salzman, “Light Scatter Analysis of Microalgae,” Biophys. J. 22, 29 (1978).

Roach, D. M.

J. R. V. Zaneveld, D. M. Roach, H. Pak, “The Determination of the Index of Refraction Distribution of Oceanic Particulates,” J. Geophys. Res. 79, 4091 (1974).

Ryther, J.

R. R. L. Guillard, J. Ryther, “Studies on Marine Planktonic Diatoms I. Cyclotella nana Hustedt and Detonula confervacea (cleve) Gran,” Can. J. Microbiol. 8, 229 (1962).

Salzman, G. C.

B. J. Price, V. H. Kollman, G. C. Salzman, “Light Scatter Analysis of Microalgae,” Biophys. J. 22, 29 (1978).

G. C. Salzman, P. F. Mullaney, B. J. Brice, “Light Scattering Approaches to Cell Characterization,” in Flow Cytometry and Sorting, M. R. Melamed, P. F. Mullaney, M. L. Mendelsohn, Eds. (Wiley, New York, 1979).

Sato, M.

Selvin, R.

E. Haugen, R. Selvin, Assistant Curator of the Center for Culture of Marine Phytoplankton; personal communication.

Sheldon, R. W.

R. W. Sheldon, T. R. Parsons, A Practical Manual on the Use of the Coulter Counter in Marine Science (Coulter Electronics Sales Co., Toronto, 1967).

Sieburth, J. M.

M. E. Sieracki, P. W. Johnson, J. M. Sieburth, “Detection, Enumeration and Sizing of Planktonic Bacteria by Image Analyzed Epifluorescence Microscopy,” Appl. Environ. Microbiol. 49, 799 (1985).

Sieracki, M. E.

M. E. Sieracki, P. W. Johnson, J. M. Sieburth, “Detection, Enumeration and Sizing of Planktonic Bacteria by Image Analyzed Epifluorescence Microscopy,” Appl. Environ. Microbiol. 49, 799 (1985).

Takamura, T.

M. Tanaka, T. Nakajima, T. Takamura, “Simultaneous Determination of Complex Refractive Index and Size Distribution of Airborne and Water Suspended Particles from Light Scattering Measurements,” J. Meteorol. Soc. Jpn. 60, 1259 (1982).

Tanaka, M.

M. Tanaka, T. Nakajima, T. Takamura, “Simultaneous Determination of Complex Refractive Index and Size Distribution of Airborne and Water Suspended Particles from Light Scattering Measurements,” J. Meteorol. Soc. Jpn. 60, 1259 (1982).

Tomlinson, R. D.

K. L. Carder, R. D. Tomlinson, G. F. Beardsley, “A Technique for the Estimation of the Indices of Refraction of Marine Phytoplankters,” Limnol. Oceanogr. 17, 833 (1972).

Trask, B. J.

B. J. Trask, G. J. Van Den Engh, J. H. B. W. Elgershuizen, “Analysis of Phytoplankton by Flow Cytometry,” Cytometry 2, 258 (1982).

Tycko, D. H.

Van Den Engh, G. J.

B. J. Trask, G. J. Van Den Engh, J. H. B. W. Elgershuizen, “Analysis of Phytoplankton by Flow Cytometry,” Cytometry 2, 258 (1982).

Yentsch, C. M.

C. M. Yentsch, C. S. Yentsch, “Emergence of Optical Instrumentation for Measuring Biological Properties,” Oceanogr. Mar. Biol. Ann. Rev. 22, 55 (1984).

C. M. Yentsch et al., “Flow Cytometry and Sorting: A Technique for Analysis and Sorting of Aquatic Particles,” Limnol. Oceanogr. 28, 1275 (1983).

Yentsch, C. S.

C. M. Yentsch, C. S. Yentsch, “Emergence of Optical Instrumentation for Measuring Biological Properties,” Oceanogr. Mar. Biol. Ann. Rev. 22, 55 (1984).

Zaneveld, J. R. V.

J. R. V. Zaneveld, D. M. Roach, H. Pak, “The Determination of the Index of Refraction Distribution of Oceanic Particulates,” J. Geophys. Res. 79, 4091 (1974).

Appl. Environ. Microbiol. (1)

M. E. Sieracki, P. W. Johnson, J. M. Sieburth, “Detection, Enumeration and Sizing of Planktonic Bacteria by Image Analyzed Epifluorescence Microscopy,” Appl. Environ. Microbiol. 49, 799 (1985).

Appl. Opt. (5)

Biophys. J. (1)

B. J. Price, V. H. Kollman, G. C. Salzman, “Light Scatter Analysis of Microalgae,” Biophys. J. 22, 29 (1978).

Can. J. Microbiol. (1)

R. R. L. Guillard, J. Ryther, “Studies on Marine Planktonic Diatoms I. Cyclotella nana Hustedt and Detonula confervacea (cleve) Gran,” Can. J. Microbiol. 8, 229 (1962).

Curr. Microbiol. (1)

J. H. Paul, W. H. Jeffrey, “Measurement of Diameters of Estuarine Bacteria and Particulates in Natural Water Samples by Use of a Submicron Particle Analyzer,” Curr. Microbiol. 10, 7 (1984).

Cytometry (1)

B. J. Trask, G. J. Van Den Engh, J. H. B. W. Elgershuizen, “Analysis of Phytoplankton by Flow Cytometry,” Cytometry 2, 258 (1982).

J. Geophys. Res. (1)

J. R. V. Zaneveld, D. M. Roach, H. Pak, “The Determination of the Index of Refraction Distribution of Oceanic Particulates,” J. Geophys. Res. 79, 4091 (1974).

J. Meteorol. Soc. Jpn. (1)

M. Tanaka, T. Nakajima, T. Takamura, “Simultaneous Determination of Complex Refractive Index and Size Distribution of Airborne and Water Suspended Particles from Light Scattering Measurements,” J. Meteorol. Soc. Jpn. 60, 1259 (1982).

Limnol. Oceanogr. (2)

K. L. Carder, R. D. Tomlinson, G. F. Beardsley, “A Technique for the Estimation of the Indices of Refraction of Marine Phytoplankters,” Limnol. Oceanogr. 17, 833 (1972).

C. M. Yentsch et al., “Flow Cytometry and Sorting: A Technique for Analysis and Sorting of Aquatic Particles,” Limnol. Oceanogr. 28, 1275 (1983).

Oceanogr. Mar. Biol. Ann. Rev. (1)

C. M. Yentsch, C. S. Yentsch, “Emergence of Optical Instrumentation for Measuring Biological Properties,” Oceanogr. Mar. Biol. Ann. Rev. 22, 55 (1984).

Other (6)

G. C. Salzman, P. F. Mullaney, B. J. Brice, “Light Scattering Approaches to Cell Characterization,” in Flow Cytometry and Sorting, M. R. Melamed, P. F. Mullaney, M. L. Mendelsohn, Eds. (Wiley, New York, 1979).

E. Haugen, R. Selvin, Assistant Curator of the Center for Culture of Marine Phytoplankton; personal communication.

R. W. Sheldon, T. R. Parsons, A Practical Manual on the Use of the Coulter Counter in Marine Science (Coulter Electronics Sales Co., Toronto, 1967).

N. G. Jerlov, Marine Optics (Elsevier, Amsterdam, 1976).

O. B. Brown, H. R. Gordon, “Tables of Mie scattering Functions for Low Index Particles Suspended in Water,” U. Miami Optical Physics Laboratory, Publication M1APW-OP-765 (1971).

Using Brown and Gordon Tables and an assumed concentration of 104 cells/mliter of 5-μm diameter and respective relative indices of refraction of 1.01 and 1.03.

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Figures (6)

Fig. 1
Fig. 1

Schematic drawing of the functional components of the EPICS V flow cytometer.

Fig. 2
Fig. 2

Photomicrographs of the calibration standards: (a) heptane; (b) nonane; (c) dodecane; (d) crown glass.

Fig. 3
Fig. 3

Light scatter data for the calibration standards on the flow cytometer. Shown are the 3-D frequency distributions of log 90° light scatter (L90LS) vs forward angle light scatter (FALS) and the 2-D contour projections of the same data. The composite plot of all four calibration standards is also shown.

Fig. 4
Fig. 4

Light scatter data for the algae samples. Two-dimensional contour plots are shown as well as the computed best-fit slopes. For each sample the lines of the hydrocarbons (from Fig. 3) are also shown. A typical histogram of fluorescence is also shown.

Fig. 5
Fig. 5

Theoretical curves of flow cytometrically measurable log 90° light scatter (L90LS) vs forward angle light scatter (FALS) for size distributions of spherical particles of relative real refractive indices 1.01, 1.03 and 1.05.

Fig. 6
Fig. 6

Calibration curve of flow cytometrically determined slope of L90LS vs FALS compared to relative real refractive index. Also shown are the measured slopes of the algae and the estimated refractive indices.

Tables (1)

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Table I Flow Cytometric Algal Light Scatter Measurements and Determined Refractive Indices

Equations (1)

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β ( θ ) = d I ( θ ) E d v ( e . g . , Jerlov 8 ) ,

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