Biological insights and technical obstacles during rapid at-line single-cell analysis of algal cultures based on label-free flow cytometry

The potential of label-free flow cytometry (FC) for bioprocess monitoring was determined for its application to model cultures of Chlamydomonas. A theoretical approach based on Lorenz-Mie theory revealed that the storage compounds within algal cells, specifically lipid bodies and starch granules, are the most important contributors to the commonly used wideangle light scattering signal in FC devices. As a result of cellular growth dynamics, a pronounced nonlinearity appeared in the FC data which resulted in distorted chlorophyll and wide-angle scatter distributions. In relation to the nonlinearity, the potential of label-free FC as a tool to follow trajectories of cellular development was determined and revealed high cellto-cell heterogeneity for cellular chlorophyll amount and the wide-angle light scattering signal, showing a spread of about one order of magnitude. Over the developmental cycle, a linear correlation was observed between mean values of the distributions of cellular chlorophyll amount and the wide-angle light scattering signal. Surprisingly, on the singlecell level a relatively low but dynamic correlation over time was observed. Additionally, the mother cells within the cell population differentiated into two subpopulations. In conclusion, nonparallel development of the algal cells was revealed, which expands the view about synchronicity in algal cultures.