Figure 1. Dynamics of V. cholerae biofilm formation.

a, Cells constitutively expressing a green fluorescent protein (sfGFP) were imaged with spinning disc confocal microscopy. Images at three different z-planes are highlighted. b, 3D reconstruction of the biofilm shown in panel a, where each cell is coloured according to the nematic order parameter $\mathit{\text{S}}=<\mathbf{3}/\mathbf{2}{({\widehat{\mathit{\text{n}}}}_{\mathit{\text{i}}}·{\widehat{\mathit{\text{n}}}}_{\mathit{\text{j}}})}^{\mathbf{2}}-\mathbf{1}/\mathbf{2}>$ in its vicinity. High time resolution (Δt = 5–10 min) imaging allowed us to track cell lineages and discriminate cells (white) which are not direct descendants of the biofilm founder cell. c, The extracellular matrix protein RbmA mediates cell-cell adhesion and is distributed throughout the biofilm, as visualized by immunofluorescence. d, Time-resolved WT* biofilm growth series. Each cell is coloured according to the cellular alignment with the z-axis (for the ΔrbmA mutant see Supplementary Fig. 6). e-f, Heatmaps showing spatially resolved single-cell measurements of different biofilm structural properties inside WT* (e) and ΔrbmA (f) biofilms, which are used to characterize biofilm formation (n > 3 biofilms, standard deviations are shown in Supplementary Figs. 5 and 7 and the differences among both strains are highlighted in Supplementary Fig. 8), as a function of the distance to the biofilm centre (d_centre) and the number of cells inside the biofilm (N_cells).