Our current understanding of biofilm architecture stems almost entirely from coarse-grained images of the overall, opaque, mature biofilm conglomerate. To peak into biofilms, we developed a microscopy technique that combines improved optics, imaging conditions, and advanced analysis algorithms to enable single-cell resolution throughout the maturation of living bacterial biofilms – from one founder cell to ~10,000 cells. Using V. cholerae as a model biofilm former, our work revealed surprising, precise cell ordering inside biofilms. Cells at the center of a mature biofilm dynamically align in a nematic order perpendicular to the surface, just like liquid crystal molecules in TV displays. By combining mutagenesis and in situ immunostaining of different matrix components, we traced the origin of cell ordering to a competition between cell proliferation, cell-to-surface adhesion, and cell-to-cell adhesion (Yan et al. 2016).
Movie 1: Growth of V. cholerae biofilm from one founder cell to 8,500 cells. Imaging began 1 h after attachment; total duration is 20 h with 30-min time steps. Cross-sectional images of the bottom cell layer are shown.
In collaboration with theoretical physicists, we developed agent-based computer simulations to investigate cell-surface interactions (Beroz et al., 2018). A biofilm starts growing on a surface as a one-cell-layer thick 2D film. During expansion, cells experience increasing mechanical pressure as they divide and push against their neighbors. Ultimately, the pressure from pushing exceeds the cell-to-surface adhesion force and causes individual cells to reorient vertically. When verticalized cells divide, they place their offspring further into the third dimension, thus the biofilm gradually transitions from 2D to 3D. By changing cell lengths with chemicals, we can control the timing of verticalization and therefore the overall biofilm shape.
Movie 2: Simulated biofilm growth. Starting from a founder cell that lies horizontal to the surface, we simulated the growth of a biofilm cluster without (Left) and with (Right) cell-to-surface adhesion.
Currently, we are investigating how such anisotropic cellular-scale architecture affects local biofilm material properties and gene expression. We will combine live imaging with in situ mechanical measurements to simultaneously follow the development of biofilm structures and the corresponding mechanics as a function of time. We are also applying the single-cell imaging technique to other biofilm forming species to ask whether the cellular ordering observed in V. cholerae is generalizable to other biofilm-forming species.