High throughput imaging to the diatom Cyclotella cryptica demonstrates substantial cell-to-cell variability in the rate and extent of triacylglycerol accumulation

• Cyclotella cryptica over accumulates TAG under both Si and N deprivation.
• There are two distinct rates of TAG accumulation.
• Imaging flow cytometry provides a novel method for characterizing TAG accumulation.
• Intrapopulation heterogeneity is a phenomenon that is prevalent during stress.

In microalgal cultures, most analyses of cellular processes are done in bulk, on the entire population of cells. Information gained from this is representative of the mean; however, it obscures the richness of cell-to-cell variation, which is a well-documented phenomenon. Using imaging flow cytometry, we evaluate changes in triacylglycerol (TAG) content and chlorophyll resulting from silicon or nitrogen deprivation in the diatom Cyclotella cryptica. This approach allows detailed interrogation of large numbers of individual cells and reveals cell-to-cell variation. This study demonstrates several previously undescribed phenomena related to TAG accumulation in microalgae. First, the rate of TAG accumulation varies over time, with a faster rate occurring at the latter stage of the process, resulting in hyperaccumulation in which the majority of the cell volume is comprised of lipid droplets. In C. cryptica and other diatoms this hyperaccumulation occurs strictly under autotrophic conditions. Second, there are distinct responses to silicon or nitrogen limitation, and variation within a given type of limitation treatment. Under most conditions there is a large spread in the population when measuring either chlorophyll or TAG. Heterogeneity within the total population indicates that caution should be taken in interpreting bulk measurements for a variety of variables (TAG, transcript, protein, metabolites, etc.) related to cellular responses. However, a potential means to couple subpopulation-level responses with bulk analysis approaches is described, which could take advantage of the nuances observed during the TAG accumulation process.