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Applied Optics

Applied Optics


  • Vol. 42, Iss. 3 — Jan. 20, 2003
  • pp: 526–541

Flow cytometric determination of size and complex refractive index for marine particles: comparison with independent and bulk estimates

Rebecca E. Green, Heidi M. Sosik, Robert J. Olson, and Michele D. DuRand  »View Author Affiliations

Applied Optics, Vol. 42, Issue 3, pp. 526-541 (2003)

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We advance a method to determine the diameter D and the complex refractive index (n + n′i) of marine particles from flow cytometric measurements of forward scattering, side scattering, and chlorophyll fluorescence combined with Mie theory. To understand better the application of Mie theory with its assumptions to flow cytometry (FCM) measurements of phytoplankton cells, we evaluate our flow cytometric-Mie (FCM-Mie) method by comparing results from a variety of phytoplankton cultures with independent estimates of cell D and with estimates of n and n′ from the inversion of bulk measurements. Cell D initially estimated from the FCM-Mie method is lower than independent estimates, and n and n′ are generally higher than bulk estimates. These differences reflect lower forward scattering and higher side scattering for single-cell measurements than predicted by Mie theory. The application of empirical scattering corrections improves FCM-Mie estimates of cell size, n, and n′; notably size is determined accurately for cells grown in both high- and low-light conditions, and n′ is correlated with intracellular chlorophyll concentration. A comparison of results for phytoplankton and mineral particles suggests that differences in n between these particle types can be determined from FCM measurements. In application to natural mixtures of particles, eukaryotic pico/nanophytoplankton and Synechococcus have minimum mean values of n′ in surface waters, and nonphytoplankton particles have higher values of n than phytoplankton at all depths.

© 2003 Optical Society of America

OCIS Codes
(010.4450) Atmospheric and oceanic optics : Oceanic optics
(120.4640) Instrumentation, measurement, and metrology : Optical instruments
(260.2510) Physical optics : Fluorescence
(290.4020) Scattering : Mie theory
(290.5850) Scattering : Scattering, particles
(350.4990) Other areas of optics : Particles

Original Manuscript: May 21, 2002
Revised Manuscript: October 23, 2002
Published: January 20, 2003

Rebecca E. Green, Heidi M. Sosik, Robert J. Olson, and Michele D. DuRand, "Flow cytometric determination of size and complex refractive index for marine particles: comparison with independent and bulk estimates," Appl. Opt. 42, 526-541 (2003)

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  1. A. Morel, L. Prieur, “Analysis of variations in ocean color,” Limnol. Oceanogr. 22, 709–722 (1977). [CrossRef]
  2. J. T. O. Kirk, Light and Photosynthesis in Aquatic Ecosystems (Cambridge University, New York, 1983).
  3. M. R. Lewis, J. J. Cullen, “From cells to the ocean: satellite ocean color,” in Particle Analysis in Oceanography, S. Demers, ed. (Springer-Verlag, New York, 1991), pp. 325–337. [CrossRef]
  4. D. Stramski, A. Bricaud, A. Morel, “Modeling the inherent optical properties of the ocean based on the detailed composition of the planktonic community,” Appl. Opt. 40, 2929–2945 (2001). [CrossRef]
  5. R. J. Olson, E. R. Zettler, M. D. DuRand, “Phytoplankton analysis using flow cytometry,” in Aquatic Microbial Ecology, P. F. Kemp, B. F. Shen, E. B. Shen, J. J. Cole, eds. (Lewis Publishers, Boca Raton, Fla., 1993), pp. 175–186.
  6. D. Marie, F. Partensky, S. Jacquet, D. Vaulot, “Enumeration and cell cycle analysis of natural populations of marine picoplankton by flow cytometry using the nucleic acid stain SYBR Green I,” Appl. Environ. Microbiol. 63, 186–193 (1997). [PubMed]
  7. R. J. Olson, E. R. Zettler, O. K. Anderson, “Discrimination of eukaryotic phytoplankton cell types from light scatter and autofluorescence properties measured by flow cytometry,” Cytometry 10, 636–643 (1989). [CrossRef] [PubMed]
  8. M. D. DuRand, “Phytoplankton growth and diel variations in beam attenuation through individual cell analysis,” Ph.D. dissertation (Massachusetts Institute of Technology/Woods Hole Oceanographic Institution, Woods Hole, Mass., 1995).
  9. K. Y. H. Gin, S. W. Chisholm, R. J. Olson, “Seasonal and depth variation in microbial size spectra at the Bermuda Atlantic time series station,” Deep-Sea Res. I 46, 1221–1245 (1999). [CrossRef]
  10. K. K. Cavender-Bares, “Size distributions, population dynamics, and single-cell properties of marine plankton in diverse nutrient environments,” Ph.D. dissertation (Massachusetts Institute of Technology, Cambridge, Mass., 1999).
  11. R. K. Y. Chan, K. M. Un, “Real-time size distribution, concentration, and biomass measurement of marine phytoplankton with a novel dual-beam laser fluorescence Doppler cytometer,” Appl. Opt. 40, 2956–2965 (2001). [CrossRef]
  12. S. G. Ackleson, R. W. Spinrad, “Size and refractive index of individual marine particulates: a flow cytometric approach,” Appl. Opt. 27, 1270–1277 (1988). [CrossRef] [PubMed]
  13. C. D. Mobley, Light and Water; Radiative Transfer in Natural Waters (Academic, San Diego, Calif., 1994).
  14. M. J. Perry, S. M. Porter, “Determination of the cross-section absorption coefficient of individual phytoplankton cells by analytical flow cytometry,” Limnol. Oceanogr. 34, 1727–1738 (1989). [CrossRef]
  15. A. Bricaud, A. L. Bedhomme, A. Morel, “Optical properties of diverse phytoplanktonic species: experimental results and theoretical interpretation,” J. Plankton Res. 10, 851–873 (1988). [CrossRef]
  16. M. D. DuRand, R. J. Olson, “Diel patterns in optical properties of the chlorophyte Nannochloris sp.: relating the individual cell to bulk measurements,” Limnol. Oceanogr. 43, 1107–1118 (1998). [CrossRef]
  17. H. Volten, J. F. de Haan, J. W. Hovenier, R. Schreurs, W. Vassen, A. G. Dekker, H. J. Hoogenboom, F. Charlton, R. Warts, “Laboratory measurements of angular distributions of light scattered by phytoplankton and silt,” Limnol. Oceanogr. 43, 1180–1197 (1998). [CrossRef]
  18. S. G. Ackleson, D. B. Robins, J. A. Stephens, “Distributions in phytoplankton refractive index and size within the North Sea,” in Ocean Optics IX, S. G. Ackleson, ed., Proc. SPIE925, 317–325 (1988). [CrossRef]
  19. W. S. Pegau, D. Gray, J. R. V. Zaneveld, “Absorption and attenuation of visible and near-infrared light in water: dependence on temperature and salinity,” Appl. Opt. 36, 6035–6046 (1997). [CrossRef] [PubMed]
  20. R. A. Reynolds, D. Stramski, D. A. Kiefer, “The effect of nitrogen limitation on the absorption and scattering properties of the marine diatom Thalassiosira pseudonana,” Limnol. Oceanogr. 42, 881–892 (1997). [CrossRef]
  21. D. Stramski, R. A. Reynolds, “Diel variations in the optical properties of a marine diatom,” Limnol. Oceanogr. 38, 1347–1364 (1993). [CrossRef]
  22. S. W. Jeffrey, N. A. Welschmeyer, “Spectrophotometric and fluorometric equations in common use in oceanography,” in Phytoplankton Pigments in Oceanography: Guidelines to Modern Methods, S. W. Jeffrey, R. F. C. Mantoura, S. W. Wright, eds. (UNESCO, Paris, 1997), pp. 597–615.
  23. S. W. Wright, S. W. Jeffrey, R. F. C. Mantoura, “Evaluation of methods and solvents for pigment extraction,” in Phytoplankton Pigments in Oceanography, S. W. Jeffrey, R. F. C. Mantoura, S. W. Wright, eds. (UNESCO, Paris, 1997), pp. 261–282.
  24. A. Shalapyonok, R. J. Olson, L. S. Shalapyonok, “Arabian Sea phytoplankton during Southwest and Northeast Monsoons 1995: composition, size structure and biomass from individual cell properties measured by flow cytometry,” Deep-Sea Res. II 48, 1231–1262 (2001). [CrossRef]
  25. M. D. DuRand, R. E. Green, H. M. Sosik, R. J. Olson, “Diel variations in optical properties of Micromonas pusilla (Prasinophyceae),” J. Phycol. 38, 1132–1142 (2002). [CrossRef]
  26. R. R. L. Guillard, “Culture of phytoplankton for feeding marine invertebrates,” in Culture of Marine Invertebrate Animals, W. L. Smith, M. H. Chanely, eds. (Plenum, New York, 1975), pp. 29–60. [CrossRef]
  27. T. D. Dickey, A. J. Williams, “Interdisciplinary ocean process studies on the New England shelf,” J. Geophys. Res. 106, 9427–9434 (2001). [CrossRef]
  28. H. M. Sosik, R. E. Green, W. S. Pegau, C. S. Roesler, “Temporal and vertical variability in optical properties of New England shelf waters during late summer and spring,” J. Geophys. Res. 106, 9455–9472 (2001). [CrossRef]
  29. H. C. van de Hulst, Light Scattering by Small Particles (Wiley, New York, 1957).
  30. A. Bricaud, A. Morel, “Light attenuation and scattering by phytoplankton cells: a theoretical modeling,” Appl. Opt. 25, 571–580 (1986). [CrossRef]
  31. C. F. Bohren, D. R. Hoffman, Absorption and Scattering of Light by Small Particles (Wiley, New York, 1983).
  32. D. Stramski, “Refractive index of planktonic cells as a measure of cellular carbon and chlorophyll a content,” Deep-Sea Res. I 46, 335–351 (1999). [CrossRef]
  33. S. G. Ackleson, D. B. Robins, “Flow cytometric determinations of North Sea phytoplankton optical properties,” Netherlands J. Sea Res. 25, 11–18 (1990). [CrossRef]
  34. M. D. DuRand, R. J. Olson, S. W. Chisholm, “Phytoplankton population dynamics at the Bermuda Atlantic Time-series station in the Sargasso Sea,” Deep-Sea Res. II 48, 1983–2003 (2001). [CrossRef]
  35. R. J. Olson, S. W. Chisholm, E. R. Zettler, M. A. Altabet, J. A. Dusenberry, “Spatial and temporal distributions of prochlorophyte picoplankton in the North Atlantic Ocean,” Deep-Sea Res. 37, 1033–1051 (1990). [CrossRef]
  36. M. S. Twardowski, E. Bass, J. B. Macdonald, W. S. Pegau, A. H. Barnard, J. R. Zaneveld, “A model for estimating bulk refractive index from the optical backscattering ratio and the implications for understanding particle composition in case I and case II waters,” J. Geophys. Res. 106, 14129–14142 (2001). [CrossRef]
  37. R. P. Bukata, J. H. Jerome, K. Y. Kondryatev, D. V. Pozdnyakov, Optical Properties and Remote Sensing of Inland and Coastal Waters (CRC Press, Boca Raton, Fla., 1995).
  38. B. G. Mitchell, D. A. Kiefer, “Chlorophyll a specific absorption and fluorescence excitation spectra for light-limited phytoplankton,” Deep-Sea Res. 35, 639–663 (1988). [CrossRef]
  39. H. M. Sosik, S. W. Chisholm, R. J. Olson, “Chlorophyll fluorescence from single cells: interpretation of flow cytometric signals,” Limnol. Oceanogr. 34, 1749–1761 (1989). [CrossRef]
  40. H. M. Sosik, B. G. Mitchell, “Absorption, fluorescence and quantum yield for growth in nitrogen limited Dunaliella tertiolecta,” Limnol. Oceanogr. 36 (5), 910–921 (1991). [CrossRef]
  41. K. Witkowski, T. Krol, M. Lotocka, “The light scattering matrix of Chlorella vulgaris cells and its variability due to cell modification,” Oceanologia 36, 19–31 (1994).
  42. K. Witkowski, T. Król, A. Zieliński, E. Kuteń, “A light-scattering matrix for unicellular marine phytoplankton,” Limnol. Oceanogr. 43, 859–869 (1998). [CrossRef]

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