Cryo-SEM Reveals Native Architecture and Matrix Complexity in P. aeruginosa Biofilms

Pseudomonas aeruginosa biofilms pose a critical challenge in clinical settings due to their high resistance to antibiotics and immune clearance, primarily attributed to the protective extracellular matrix (ECM). Accurate visualization of biofilm structure in its native state is crucial for understanding its resilience and guiding the development of targeted therapeutics. We used three different sample preparation methods - critical-point drying (CPD), air-drying (AD), and high-pressure cryogenic freezing for the sample preparation of native biofilms grown on solid substrates and microcolonies grown within alginate matrices. Our goal was to compare their morphologies and spatial organizations using SEM and cryogenic SEM (cryo-SEM). Cryo-SEM revealed Pseudomonas biofilms as highly hydrated, organized structures, preserving delicate ECM features and cellular morphology. The ECM and cell distributions were severely distorted in CPD and air-dried samples due to dehydration. Notably, cryo-SEM revealed that mucoid P. aeruginosa grown for 4 days is spatially separated and short-range ordered in the extensive ECM with nearest-neighbor distances averaging 1.26 µm, while PAO1 showed a broader distribution in nearest-neighbor distances with an average of 1.64 µm, both challenging the view of biofilms as closely packed cell aggregates. Furthermore, distinct differences in cryo-SEM-visualized ECM structure were observed between PAO1 and mucoid strains. The latter formed a voluminous, filamentous matrix rich in aligned fibrillar ultrastructures, which likely derive from protein ECM components. In contrast, PAO1 displayed denser biofilms with mesh-like ECMs and higher cell densities. The cell densities of both biofilms were observed to be lowest close to the substrate. While PAO1 displays a monotonic increase in cell density from the substrate to the biofilm surface, mucoid biofilms displayed a stratified pattern, with significantly higher densities at the surface and mid-depth than near the substrate. Our findings establish cryo-SEM as a superior method for high-fidelity imaging of biofilms, offering new insights into the structural organization of cells and ECM in P. aeruginosa biofilms.