Cryo-SEM Reveals Native Architecture and Matrix Complexity in P. aeruginosa Biofilms
Osondu-Chuka, G. O.; Schandl, S.; Subbiahdoss, G.; Ovsianikov, A.; Guillaume, O.; Reimhult, E.
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Pseudomonas aeruginosa biofilms resist antibiotics and immune clearance through their biofilm organization, yet their native spatial organization and extracellular matrix (ECM) remain difficult to resolve. We compare air-drying, critical-point drying, and high-pressure cryogenic freezing for preparing P. aeruginosa biofilms for scanning electron microscopy (SEM) imaging. Only cryo-SEM preserved hydrated ECM ultrastructure, cellular morphology, and organization, whereas other methods caused severe distortions due to dehydration. Combining cryo-SEM with confocal laser scanning microscopy (CLSM) enabled quantitative spatial analyses with high resolution across large biofilm volumes. Nearest-neighbor distances, radial distribution functions, and Ripleys H-functions revealed broadly distributed cell spacings with a preferred distance of [~]1 {micro}m and only weak clustering in 4-day-old biofilms, challenging the view of biofilms as densely packed microcolonies. Mucoid and PAO1 strains exhibited distinct ECM ultrastructures and pronounced vertical stratification. This multiscale approach establishes cryo-SEM and CLSM as complementary tools for quantitative, high-fidelity biofilm analysis.
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