Quantifying motility-based biological responses in Chlamydomonas reinhardtii using an integrated single-cell framework

Quantitative monitoring of motility in biological microswimmers is an essential instrument for understanding how microorganisms perceive, process, and translate external stimuli into dynamic behavioural responses. However, tactical responses emerge from complex biophysical mechanisms that operate simultaneously at the individual and collective levels, giving rise to intra-population heterogeneity that can be difficult to quantify with population-averaged approaches: when the trajectories of kinematically distinct subgroups are aggregated, coherent responses may appear weak or confused even in the presence of organised subpopulations. In this work, we present a single-cell tracking-based method for the rigorous kinematic characterisation of the phototactic response of Chlamydomonas reinhardtii , with the aim of resolving behavioural variability and its temporal evolution within heterogeneous populations. The workflow integrates automated large-scale tracking, detailed trajectory reconstruction, single-cell and population-level kinematic analysis, segmentation into coherent behavioural subpopulations (stationary, confined/circling, and directional) via clustering in circular feature space, and characterisation of dynamic motion regimes through time-windowed mean squared displacement (MSD) analysis. By explicitly decomposing the population into coherent kinematic components and quantifying their relative contributions, the method provides “behavioural resolution” of mixed phototactic responses. Based exclusively on open-source software and robust mathematical analysis, the method is highly reproducible and easily extendable to the study of other biological or synthetic microswimmers subjected to controlled external stimuli.