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Biofilms are multicellular microbial communities that encase themselves in an extracellular matrix (ECM) of secreted biopolymers and attach to surfaces and interfaces. Bacterial biofilms are detrimental in hospital and industrial settings, but they can be beneficial, for example, in agricultural as well as in food technology contexts. An essential property of biofilms that grants them with increased survival relative to planktonic cells is phenotypic heterogeneity, the division of the biofilm population into functionally distinct subgroups of cells. Phenotypic heterogeneity in biofilms can be traced to the cellular level; however, the molecular structures and elemental distribution across whole biofilms, as well as possible linkages between them, remain unexplored. Mapping X-ray diffraction across intact biofilms in time and space, we revealed the dominant structural features in Bacillus subtilis biofilms, stemming from matrix …