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

Abstract

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.

Contributions of phytoplankton and other particles to inherent optical properties in New England continental shelf waters

“…FCM-Mie method-A combination of FCM measurements and Mie theory was used to determine individual particle properties (referred to as the ''FCM-Mie method''; for details see Green et al 2003). We applied the FCM-Mie method to the analysis of eukaryotic phytoplankton, Synechococcus, heterotrophic prokaryotes, and nonphytoplankton in each sample.…”

“…We applied the FCM-Mie method to the analysis of eukaryotic phytoplankton, Synechococcus, heterotrophic prokaryotes, and nonphytoplankton in each sample. As described in Green et al (2003), to apply Mie theory to FCM measurements, an optimization was performed between theory and measurements using calibration particles of known D, n, and nЈ. Two different optimizations were used, one for phytoplankton and nonphytoplankton particles and a second for heterotrophic prokaryotes for which FCM settings were changed to enumerate smaller particles (Green 2002).…”

“…As described in Green et al (2003), to apply Mie theory to FCM measurements, an optimization was performed between theory and measurements using calibration particles of known D, n, and nЈ. Two different optimizations were used, one for phytoplankton and nonphytoplankton particles and a second for heterotrophic prokaryotes for which FCM settings were changed to enumerate smaller particles (Green 2002). FCM-Mie analysis was limited to particles of less than 10 m in diameter, because solutions are often not unique for larger particles.…”

“…FCM-Mie analysis was limited to particles of less than 10 m in diameter, because solutions are often not unique for larger particles. As well, an FLS and SSC correction was applied to measurements for phytoplankton cells to account for deviations of cells from the Mie theory assumptions that particles are spherical and homogenous (Green et al 2003). Particle properties (D, n, and nЈ) and optical cross-sections for absorption ( a ), scattering ( b ), and backscattering ( bb ) at 488 nm were determined from the FCM-Mie method by comparison of FCM FLS, SSC, and CHL to values in a Mie-based lookup table.…”

“…Flow cytometry has been used to enumerate and distinguish specific groups of particles, and advances have been made toward determining distributions of particle D, n, and nЈ with application to determining particle contributions to IOPs (Ackleson and Spinrad 1988;Olson et al 1989;Perry and Porter 1989;Olson et al 1993;Marie et al 1997;DuRand and Olson 1998;Claustre et al 1999;Green et al 2003).…”