Phytoplankton biovolume estimation with conventional flow cytometry

Flow cytometry is widely used to assess phytoplankton populations, yet opportunities remain to improve and simplify the extraction of ecologically meaningful cellular properties such as biovolume. We assessed the relationship between microscopy measurements and flow cytometry signals in phytoplankton samples and calibration beads to estimate biovolume and validate the extent to which “conventional” (i.e., medical) flow cytometers can be used to measure biovolume. Microscopy-based biovolume showed a strong correlation with flow cytometry signals and was best described by a forward scatter-area (FSC-A) power-law model ( R 2 = 0.89, p < 0.01). FSC-A was therefore used to define the biovolume–flow cytometry relationship for phytoplankton cells. This relationship was consistent with that observed between calibration bead volume and FSC-A; however, both cells and beads ≤2 µm in diameter (4.19 µm 3 ) deviated from the main correlation. Cell morphology did not significantly affect the FSC-A–biovolume model but did influence bead-based biovolume predictions. This study presents a simple approach for estimating phytoplankton biovolume using widely accessible medical flow cytometers and demonstrates that calibration beads provide reliable biovolume estimates for cells >5 µm 3 . As biovolume is central to phytoplankton ecology and ocean biogeochemical fluxes, this approach enhances the utility of flow cytometry for biovolume assessment.