To measure single-cell microbial activity and sub- strate utilization patterns in environmental systems, we employ a new technique using stable isotope labelling of microbial populations with heavy water (a passive tracer) and 15 N ammonium in combination with multi-isotope imaging mass spectrometry. We demonstrate simultaneous NanoSIMS analysis of hydrogen, carbon and nitrogen at high spatial and mass resolution, and report calibration data linking single-cell isotopic compositions to the correspond- ing bulk isotopic equivalents for Pseudomonas aeruginosa and Staphylococcus aureus. Our results show that heavy water is capable of quantifying in situ single-cell microbial activities ranging from gen- erational time scales of minutes to years, with only light isotopic incorporation (∼0.1 atom % 2 H). Apply- ing this approach to study the rates of fatty acid biosynthesis by single cells of S. aureus growing at different rates in chemostat culture (∼6 h, 1 day and 2 week generation times), we observe the greatest ana- bolic activity diversity in the slowest growing popu- lations. By using heavy water to constrain cellular growth activity, we can further infer the relative con- tributions of ammonium versus amino acid assimilation to the cellular nitrogen pool. The approach described here can be applied to disentangle indi- vidual cell activities even in nutritionally complex environments.
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