Figure 1. P. aeruginosa biofilms are surrounded by psl.

(A) C57BL6/N mice were infected with 1.10⁵ cfu/eye P. aeruginosa PAO1 expressing CrdA-GFP. The intravital microscopy was carried out at early (12h) and late (24h) post-infectious challenge. Individual mice were imaged longitudinally. Areas covered with bacterial microcolonies or biofilms were calculated using Image J analysis. Each symbol represents individual bacterial cluster/microcolony. Student’s t-test. (B) 3D-reconstructions of early and late bacterial biofilms. Volumes of biofilms per image were shown for comparison. (C) Biofilms were stained with fluorescently labeled anti-Psl mAb Cam003 (red) which was applied topically 15 min before imaging. Images were recorded at 40x. Psl (red) staining was adjacent or co-localized to P. aeruginosa (green). All images are representative of five individual experiments with 1–2 imaged mice per experiment. Imaging across central cornea revealed that bacterial microaggregates were seen during early hours of infection, whereas bacterial biofilms were formed as infection progressed. P. aeruginosa PAO1 CdrA-GFP (green) clusters were surrounded by red Psl-stained polysaccharide, consistent with the definition of a biofilm.