Fig. 4

Scanning transmission x-ray spectromicroscopy at 87 K of frozen-hydrated MS4 and MS11 biofilms. A Protein map of an MS11 small filament (700 nm ± 120 nm in diameter), S⁰ granules are pointed by white arrows. B Extracellular S⁰ granules (~ 300 to 850 nm in diameter) and MS11 cells imaged at 288.2 eV (amide group in proteins) and corresponding (C) protein map. D Protein map of an MS4 filament (1.46 ± 0.16 µm in diameter) with a surface-attached cell (1.9 ± 0.21 µm, red arrow). E Carbon K-edge NEXAFS spectra of filamentous bacteria (S.⁰ granule-free areas) exhibiting a major peak at 288.2 eV and other peaks mainly associated with nucleic acids (see Table S2). Spectra of MS11 and MS4 cells (red arrow) exhibit a main peak at 288.2 eV (peptide bond), and associated extracellular polymeric substances (EPS, circled in blue) show a main peak at 288.7 eV (carboxyl groups in acidic polysaccharides). See Table S2 for further details. Dashed line is at 288.2 eV. The intensity scale corresponds to the optical density. Scale bars: 1 µm (A–C) and 2 µm (D)