s were frozen at −80 °C. After storage, they were washed twice with PBS and preserved in PBS/ethanol (1:1 [v/v]) at −20 °C until analysis.
Cell enumeration and selective sorting onto the membrane
To efficiently analyze substrate incorporation into microbial cells with NanoSIMS, cells were separated from their sediment matrix and fluorescence-activated cell sorting (FACS) was conducted to concentrate and purify cells in a small area for analysis (~0.5 mm2, Supplementary Fig. 3b)14,37. Cell separation, enumeration, and FACS were all conducted in the clean-booth and clean-room facilities at the Kochi Institute for Core Sample Research, Japan Agency for Marine-Earth Science and Technology (JAMSTEC).
Cell separation, microscopy, and sorting procedures followed the method of Morono et al.37 with modifications. Eight milliliters of fixed slurry (1/3 [v/v] sediment in ethanol-PBS solution) was mixed with the same volume of 2.5% NaCl solution, followed by centrifugation at 4500 × g for 15 min, after which the supernatant was discarded and the pellet resuspended by adding 2.5% NaCl solution to be 20 mL of sediment slurry. The sediment slurry was added with 2.5 mL each of detergent mix38 (100 mM EDTA, 100 mM sodium pyrophosphate, 1% [v/v] Tween 80) and methanol, then vigorously shaken for 60 min at 500 rpm using a Shake Master (Bio Medical Science, Tokyo, Japan). After shaking, the sediment slurry was sonicated (Bioruptor UCD-250; COSMO BIO) in an ice bath for 20 cycles of 30 s at 200 W on and 30 s off. The processed slurry was then carefully layered onto a manually layered high-density cushion solution consist of (from top) 4 mL of 30% [v/v] Nycodenz, 4 mL of 50% [v/v] nycodentz, 4 mL of 80% [v/v] Nycodentz, and 4 mL of 67% [w/v] of sodium polytungstate. Samples were centrifuged at 4000 × g for 120 min, after which the supernatant, including the high-density layer(s), was carefully removed and transferred to a separate vial. Cells in the supernatant were trapped onto an Anopore Inorganic Membrane (Anodisc, Whatmann, Kent, 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 