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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.

2025-08-28 microbiology
10.1101/2025.08.28.672782 bioRxiv
Show abstract

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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