UK), washed with TE buffer, and then stained with 100 μL of SYBR Green I staining solution. After staining for 5 min, the SYBR-stained cells were washed with 2 mL of TE buffer, and then the membrane was placed into a 50 mL centrifuge tube containing 5 mL of TE buffer. Cells were detached from the membrane by sonication at 20 W for 10–30 s using a Model UH-50 Ultrasonic Homogenizer (SMT Co. Ltd., Tokyo, Japan) and concentrated to be 1.5 mL by centrifugation at 7000 × g for 10 min and discarding 3.5 mL of the supernatant. Part (0.5 mL) of the stained cell suspension was filtered onto 0.22-μm pore size black polycarbonate membrane (Isopore GTBP02500; Millipore) and used for counting microbial cells by the fluorescence color-based discriminative cell enumeration method39,40. Cells were sorted following the flow cytometry protocol of Morono et al.37 directly from the sorter onto NanoSIMS-compatible 0.2-μm polycarbonate filters coated with indium tin oxide (ITO)14,41 and non-coated membrane (Isopore GTBP02500; Millipore). ITO coating on polycarbonate membranes (Isopore GTBP02500; Millipore) was prepared by a sputtering deposition technique at Astellatech Co. Ltd. The sorted cells on the non-coated membrane were stored at −20 °C until DNA extraction.
NanoSIMS analysis of single cell-image acquisition and data processing
Cell targets were identified by fluorescence of SYBR Green I stain and marked on NanoSIMS membranes with a laser dissection microscope (LMD6000; Leica Microsystems) for ease of rediscovery on the NanoSIMS (an example is shown in Supplementary Fig. 3b). Microbial cells that incorporated stable isotope-labeled substrates were analyzed using NanoSIMS 50L (AMETEK Co. Ltd., CAMECA BU) at the Kochi Institute for Core Sample Research, JAMSTEC. Samples on the ITO-coated polycarbonate membrane were pre-sputtered at high beam currents (30 pA/s/µm2) before measurement. The 12C−, 13C−, 12C14N−, 12C15N− and 32S− secondary ions were collected and measured in parallel at a mass resolution of 8000 that is sufficient to separate 13C− from the 12CH− and 12C15N− from 13C14N−. Samples were measured using a 1–2 pA Cs+ primary beam that was rastered over 25 × 25 µm field of a 256 × 256 pixels with a counting time of 5 ms per pixel. Recorded images and data were processed using CAMECA WinImage software and OpenMIMS plugin42 in ImageJ43 distribution of Fiji44. Different scans of each image were aligned to correct image drift during acquisition. Final images were created by adding the secondary ion counts of each recorded secondary ion from each pixel over all scans. Intracellular carbon and nitrogen uptake from stable isotope-labeled substrates was calculated by drawing regions of interest (ROI) on CN− images (recognizing cells in the images) and calculating 13C/12C and the 15N/14N ratio (calculated from the 12C15N/12C14N ratio). Instrumental mass fractionation of NanoSIMS analysis was calibrated by the conversion factor obtained by comparing carbon and nitrogen isotopic ratios of E. coli cells of varying isotopic enrichments measured at single cells with NanoSIMS and at bulk with an elemental analyzer/isotope ratio mass spectrometer (EA/IRMS, FlashEA 1112/DeltaPlus Advantage, Thermo Fisher Scientific). Concentration of bicarbonate (DIC) in the original sample determined on board7 was used to calculate substrate incorporation ratio (atom %) for bicarbonate in single cells.
Biomass and isotope calculations
Isotope incorporation data analysis and display as violin plots of the kernel density function were done using R45 with the “ggplot2”46, “cowplot”47, “ggsci”48, “scales”49 packages. The “active” cell ROIs, those incorporated 13C- and/or 15N-labeled substrates, were determined by their isotopic ratio exceeding the 99.7% confidence interval of background carbon and nitrogen isotopic abundance of polycarbonate membranes (1.24 atom % for 13C and 0.446 atom % for 15N). As measures for the rate of microbial biomass synthesis, we calculated the biomass-based specific growth rate and the substrate incorporation-based biomass generation rates.