Abstract
Compelling evidence is presented that sub-micron picoplankton shape, internal structure and orientation in combination leads to a disproportionate enhancement of differential forward scatter compared with differential side scatter when analyzed with a flow cytometer. Theoretical evidence is provided which results in an order of magnitude amplification in the forward scatter direction, with little or no change in the side scatter: this discounts the possibility of "doublets" caused by multiple particles simultaneously present in the laser beam. Observational evidence from progressively finer filtered seawater samples shows up to three orders of magnitude enhancement in the forward scatter direction and sizes of Prochlorococcus close to that reported in the literature (0.61 ± 0.17 µm). It therefore seems likely that flow cytometrically observed "bi-modal size distributions" of Prochlorococcus are instead the manifestation of intra-population differences in shape (spherical - prolate with preferential alignment) and internal structure (homogenous - heterogenous).
Highlights
In our recent extensive flow cytometric analysis [1] of the variability of natural assemblages of marine picoplankton, in particular Prochlorococcus [2] and Synechococcus [3, 4], we demonstrated that Prochlorococcus frequently showed double size distribution peaks, on average (N~104 samples) centred on 0.75±0.25 and 1.75±0.25 μm; smallest peak diameters were ≤0.65 μm in the equatorial upwelling with larger cells (~0.95 μm) in the surface layers of the tropical gyres
We have shown that the shape of sub-micron Prochlorococcus cells in concert with their internal structure and preferred angular orientation, can lead to substantial changes to the scattering patterns measured by a flow cytometer
Low sensitivity used in the forward scatter channel of a flow cytometer, doesn’t allow these sub-micron particles to be attributed the correct value of differential forward scatter
Summary
In our recent extensive flow cytometric analysis [1] of the variability of natural assemblages of marine picoplankton, in particular Prochlorococcus [2] and Synechococcus [3, 4], we demonstrated that Prochlorococcus frequently showed double size distribution peaks, on average (N~104 samples) centred on 0.75±0.25 and 1.75±0.25 μm; smallest peak diameters were ≤0.65 μm in the equatorial upwelling with larger cells (~0.95 μm) in the surface layers of the tropical gyres. The term “opto-type” was coined as the two populations had distinctive side- and forward- scatter characteristics which when inverted using Mie scattering calculations resulted in the smaller having a higher refractive index than the larger sized cells. These observations were qualitatively consistent with previously-known ecotypes [5,6,7] of Prochlorococcus with distinct adaptions to environmental factors such as light intensity, temperature and nutrient concentrations [8], as well as remarkable genetic and physiological diversity [5]. In this article we look at two potential sources of the artefact: (1) inadequacies in the particle scattering calculation assumptions and (2) possible issues caused by sub-micron particles within the flow cytometer itself
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