In this study, we cultured Chlorella vulgaris cells with a range of
lipid contents, induced via
nitrogen starvation, and characterized them via flow cytometry, with
BODIPY 505/515 as a fluorescent
lipid label, and liquid-state 1H NMR spectroscopy. In doing so, we demonstrate the utility of calibrating flow cytometric measurements of algal
lipid content using triacylglyceride (TAG, also known as
triacylglycerol or
triglyceride) content per cell as measured via quantitative 1H NMR. Ensemble-averaged fluorescence of
BODIPY-labeled cells was highly correlated with average TAG content per cell measured by bulk NMR, with a linear regression yielding a linear fit with r2 = 0.9974. This correlation compares favorably to previous calibrations of flow cytometry protocols to
lipid content measured via extraction, and calibration by NMR avoids the time and complexity that is generally required for
lipid quantitation via extraction. Flow cytometry calibrated to a direct measurement of TAG content can be used to investigate the distribution of
lipid contents for cells within a culture. Our flow cytometry measurements showed that Chlorella vulgaris cells subjected to
nitrogen limitation exhibited higher mean
lipid content but a wider distribution of
lipid content that overlapped the relatively narrow distribution of
lipid content for replete cells, suggesting that
nitrogen limitation induces
lipid accumulation in only a subset of cells. Calibration of flow cytometry protocols using direct in situ measurement of TAG content via NMR will facilitate rapid development of more precise flow cytometry protocols, enabling investigation of algal
lipid accumulation for development of more productive algal
biofuel feedstocks and cultivation protocols.