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Bacterial biofilms represent a basic form of multicellular organization that confers survival advantages to constituent cells. The sequential stages of cell ordering during biofilm development have been studied in the pathogen and model biofilm-former Vibrio cholerae How do the spatial trajectories of individual cells and the collective motions of many cells drive biofilm expansion? We developed dual-view light-sheet microscopy to investigate biofilm developmental dynamics from a founder cell to a mature three-dimensional community. Tra