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

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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.