Comparison of particle properties and biovolume estimates from FlowCam and microscopy for kettle hole phytoplankton communities

Abstract The integration of high‐throughput image–based flow cytometry into routine phytoplankton assessment requires rigorous evaluation against established microscopy techniques. As method agreement is often site‐ and species‐specific, case‐by‐case validation is essential to successfully adopt emerging methods. In this study, we evaluated the agreement between two methods, FlowCam and inverted microscopy, for the analysis of phytoplankton communities from nine small freshwater kettle holes located in an agricultural landscape. Specifically, we compared automated FlowCam–derived particle properties with cell size measurements and derived biovolume estimates obtained by classical inverted microscopy across the whole phytoplankton size range. We found good agreement between one or more automated FlowCam dimensional estimates and microscopy measurements for most phytoplankton groups. However, morphologically more complex and particularly elongated taxa showed substantial discrepancies between methods. Accordingly, dimensional measurements translated into representative biovolume estimates for morphologically simple taxa, especially using prolate spheroid approximations, were similar between both methods. Divergent measurements of FlowCam‐derived widths introduced systematic biases, mainly for cylindrical approaches of biovolume calculations based on geodesics and markedly overestimated biovolumes of filamentous taxa. The findings demonstrate that the choice of FlowCam‐derived particle properties must be morphology‐specific and validated against microscopy. Although cylindrical biovolume estimates require refinement, Legendre‐based approaches performed well, supporting the integration of FlowCam into monitoring programs and experimental studies for diverse phytoplankton communities in small water bodies like kettle holes.