biostudies-literatureBerry DÖsterreichische ForschungsförderungsgesellschaftU.S. Department of EnergyAustrian Science Fund FWFEuropean Research CouncilNatural Environment Research CouncilEngineering and Physical Sciences Research CouncilE194-203https://www.ebi.ac.uk/biostudies/studies/S-EPMC4299247biostudies-literatureUnknown112(2)Microbial communities are essential to the function of virtually all ecosystems and eukaryotes, including humans. However, it is still a major challenge to identify microbial cells active under natural conditions in complex systems. In this study, we developed a new method to identify and sort active microbes on the single-cell level in complex samples using stable isotope probing with heavy water (D2O) combined with Raman microspectroscopy. Incorporation of D2O-derived D into the biomass of autotrophic and heterotrophic bacteria and archaea could be unambiguously detected via C-D signature peaks in single-cell Raman spectra, and the obtained labeling pattern was confirmed by nanoscale-resolution secondary ion MS. In fast-growing Escherichia coli cells, label detection was already possibleProceedings of the National Academy of Sciences of the United States of AmericaTracking heavy water (D2O) incorporation for identifying and sorting active microbial cells.PMC4299247DE-AC02-05CH11231EP/M002403/1P26127-B20P 26127294343NE/M002934/1NITRICARE 294343Shterzer NDecker TWoebken DSchmid MCLee TKWagner MBerry DSchintlmeister AZhu DMizrahi IRauch IHuang WEFowler PWMader EPalatinszky MWang YGibson CMPopp JHanson BTBocklitz TfalseTracking heavy water (D2O) incorporation for identifying and sorting active microbial cells.Microbial communities are essential to the function of virtually all ecosystems and eukaryotes, including humans. However, it is still a major challenge to identify microbial cells active under natural conditions in complex systems. In this study, we developed a new method to identify and sort active microbes on the single-cell level in complex samples using stable isotope probing with heavy water (D2O) combined with Raman microspectroscopy. Incorporation of D2O-derived D into the biomass of autotrophic and heterotrophic bacteria and archaea could be unambiguously detected via C-D signature peaks in single-cell Raman spectra, and the obtained labeling pattern was confirmed by nanoscale-resolution secondary ion MS. In fast-growing Escherichia coli cells, label detection was already possible2015-01-01T00:00:00Z2015 Jan2026-04-30T23:13:23.832Z2019-03-27T01:43:41ZS-EPMC42992472555051810.1073/pnas.1420406112