A single-cell analytical technology was developed for evaluating fast-growing cultures of green algae. The main part of the single-cell analysis is an epifluorescence microscopy-based cytometric approach combined with an automated image analysis algorithm and a single-threshold discrimination procedure. The reliability of the technique in terms of object recognition, evaluating particle size, and determining chlorophyll was successfully proven via reference analyses. The microscopy technique was used to determine the size of single cells, the amount of chlorophyll, and the density of chlorophyll in a model algal culture (Acutodesmus o.). The algal cells showed unexpected heterogeneity in all single-cell parameters, and exhibited a high correlation between cell size and amount of chlorophyll but a very low correlation between cell size and chlorophyll density. For a given cell size, the cell-to-cell heterogeneity of the relative chlorophyll density showed a spread of 0.02-0.08. This points to large variations in the architecture and the physiological state of the photosynthetic apparatus in the cells. This complex situation should be considered in future systems biology approaches focusing on the relationships between biomass accumulation, photosynthetic activity, and central carbon metabolism. Graphical abstract Analysis of cell-to-cell heterogeneity obtained from microscopic images.

Microalgae bear a great potential to produce lipids for biodiesel, feed, or even food applications. To understand the still not well-known single-cell dynamics during lipid production in microalgae, a novel single-cell analytical technology was applied to study a well-established model experiment. Multidimensional single-cell dynamics were investigated with a non-supervised image analysis technique that utilizes data from epi-fluorescence microscopy. Reliability of this technique was successfully proven via reference analysis. The technique developed was used to determine cell size, chlorophyll amount, neutral lipid amount, and deriving properties on a single-cellular level in cultures of the biotechnologically promising alga Acutodesmus obliquus. The results illustrated a high correlation between cell size and chlorophyll amount, but a very low and dynamic correlation between cell size, lipid amount, and lipid density. During growth conditions under nitrogen starvation, cells with low chlorophyll content tend to start the lipid production first and the cell suspension differentiated in two subpopulations with significantly different lipid contents. Such quantitative characterization of single-cell dynamics of lipid synthesizing algae was done for the first time and the potential of such simple technology is highly relevant to other biotechnological applications and to deeper investigate the process of microalgal lipid accumulation.

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