Probing the internal micromechanical properties of Pseudomonas aeruginosa biofilms by Brillouin imaging

Characterisation of the stiffness of P. aeruginosa biofilms. Typical a widefield, b Brillouin, and c fluorescence images of a single colony taken 60？h post inoculation at a depth of 24？μm inside a 42-μm thick biofilm. d Schematic model defining the various stages of a P. aeruginosa biofilm life cycle. Compact colony (i), larger colony with softer centre (ii), and hollow colony (iii). e Brillouin shift in colonies of various sizes. Data points denote the means, and error bars the standard deviations from all pixels within the ROIs enclosing the colony. ROIs drawn by visual inspection of the corresponding widefield images. Biofilms grown under constant flow velocities of either 0.042 or 0.14？cm/s (circles and triangles, respectively). f Brillouin image cross sections at different depths inside a single colony (thickness 32？μm, taken 80？h post insoculation). g Mean values and standard deviation of the Brillouin shift at different depths, measured within the ROIs marked in f. Data points connected by lines to aid visualisation. Scale bars: 10？μ