For XRMA analysis, ~ 0.4 mL of Synechococcus culture cells were transferred to a 1.5 mL microtube and washed with 0.8 mL of cold buffered ultrapure water (pH 8) to remove salts. After centrifugation (1 min at 7000 rpm at 15 ºC), 1.1 mL supernatant was replaced by cold buffered ultrapure water and centrifuged under the same conditions. This last step was repeated twice, and 1.1 mL was carefully discarded. The concentrated cells were homogenized before transferring 6 µL onto a 3 mm TEM grid coated with formvar and evaporated carbon (Ted Pella, Redding, CA, USA). The grid was air-dried and stored in a vacuum cabinet until analysis. Cells were analyzed for elemental composition on a Hitachi SU8600 Field-Emission Scanning Electron Microscope (FE-SEM) equipped with two high-resolution (123 eV) Bruker X-Flash 7|30 Energy Dispersive Spectrometers. To avoid interference with the holder, the grids were attached to the edge of the SEM pins. XRMA spectra were obtained at 20 kV of incident energy during 90 seconds of live time. The absolute beam current was measured using a Faraday cup before each analysis session, which allowed for correcting for beam current drift during the session, and ensured an accurate reference to the calibration. Polystyrene latex spheres with mean particle size of 0.6 and 1.1 µm (Sigma LB6 and LB11) were used to calibrate carbon, and ATP (Sigma, A2383) was used to obtain the calibration constants for N and P, following Norland et al. (1995).
Cell dimensions were measured from SEM micrographs using ImageJ (version 1.54g, Abràmoff et al. 2004). Synechococcus cells exhibited rod-shaped to elongated-oval morphologies. As a result, biovolume was calculated assuming a cylindrical shape with two hemispherical ends (Sun & Liu, 2003), using the width (cross section) and length (transapical section) measurements obtained for each cell in ImageJ.
