Finder grids

For cryo-EM grid preparation of purified protein, 2.5 µL of the specimen was applied to a freshly glow-discharged Quantifoil R2/2 Cu/Rh 200 mesh grid, adsorbed for 60 s, blotted for 4 to 5 s, and plunge-frozen into liquid ethane in a Vitrobot Mark IV (ThermoFisher), while the blotting chamber was maintained at 100% humidity at 10 °C. Grids for cryo-FIB milling of D. radiodurans cells were prepared as described previously (10 (link)). Briefly, D. radiodurans strain BAA-816 (obtained from the American Type Culture Collection) was grown aerobically in TGY liquid medium (49 (link)). Cells were grown for 24 h at 30°C prior to harvesting and staining with FM4-64 fluorescent membrane dye (Invitrogen). Four microliter of cells was loaded on Finder grids (Electron Microscopy Sciences) and plunge-frozen in a liquid ethane–propane mixture kept at liquid nitrogen temperatures using a Vitrobot Mark IV (Thermo Fisher Scientific). Grids were clipped and stored under liquid nitrogen.

Tissue samples were fixed for 12 h at 4 °C, rinsed three times with PHEM buffer (1.5× PHEM and 9 % (w/v) sucrose) and post fixed for 1.5 h with 1 % (w/v) osmium tetroxide in Milli-Q water. Samples were dehydrated in a graded series of ethanol and embedded in EPON Araldite. Embedded tissue was sectioned perpendicular to the surface of the sponge. Ultrathin (120 nm) and semithin (500 nm) sections were cut using a Reichert Ultracut S microtome. Ultrathin sections were transferred to finder grids (Electron Microscopy Sciences, Hatfield, PA, USA), stained with uranyl acetate and lead citrate, and imaged at 100 kV accelerating voltage using a Philips CM10 transmission electron microscope (TEM). These high-resolution images provided an initial characterisation of the tissue structure of both sponges, particularly regarding symbiont density and location (Fig. 1b, e). Semithin sections were transferred to silicon wafers, stained as above, and imaged with a Zeiss Sigma field emission scanning electron microscope (SEM) at 8 kV. Electron microscopy was performed at the Electron Microscopy Centre Amsterdam (EMCA). Regions of interest were identified by SEM and sample maps made to guide NanoSIMS analysis. One replicate per species and food source from incubations at T₀, T_0.25, T_0.5, T₃, and T₄₈ was selected for NanoSIMS analysis.