Calcification state of coccolithophores can be assessed by light scatter depolarization measurements with flow cytometry

Coccolithophores are important primary producers and a dominant group of calcifying organisms in the ocean. Calcification state depends on genetic, physiological and environmental factors. We show that flow cytometric measurement of the depolarization forward scattered light using a Brewster's Window Analyzer can be used to quantify the degree of calcification of coccolithophores at the single-cell level. Calcite-containing particles or cells were distinguished from non-calcified particles or cells by high values of forward scatter light with polarization orthogonal to that of the laser. Forward scatter polarization state varied strongly and linearly with the number of attached coccoliths per coccosphere when Emiliania huxleyi cells were first completely decalcified and then allowed to rebuild coccospheres. Cells of the heavily calcified E. huxleyi R-morphotype strain NZEH were also grown in different extracellular Ca2+ concentrations, forming complete coccospheres that contained similar numbers of attached coccoliths but varied in total calcite mass. Forward scatter polarization state varied strongly and linearly with coccosphere calcite mass. In contrast, forward scatter polarization state of detached coccoliths did not vary significantly with calcite weight, although forward scatter and side scatter did. Treatments had relatively minor effects on forward scatter, side scatter and forward scatter polarization state of decalcified cells, suggesting that depolarization of forward scatter light from E. huxleyi cells might be linearly determined, to a first approximation, by the ratio of surface calcite to organic protoplast. We suggest that flow cytometric measurement of forward scatter depolarization provides a potentially valuable method for analysis of calcification state of individual cells.