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

Abstract

Pseudomonas aeruginosa (PA) biofilms resist antibiotics and immune clearance through their multicellular community organization. Yet, the native spatial arrangement of cells and the extracellular matrix (ECM) remain poorly understood, as research has largely focused on genetics and regulatory networks rather than physical structure. This has limited our understanding of how physical structural organization connects to biofilm function. Here, we combine high-pressure-freezing cryo-SEM, depth-resolved confocal microscopy, and quantitative spatial analysis to examine 4-day-old mucoid and PAO1 biofilms in a near-native state. In contrast to the dense cellular aggregates often inferred from dehydrated samples, cryo-SEM revealed bacterial cells as individually embedded within a continuous extracellular matrix. Spatial statistics revealed a preferred intercellular spacing of approximately 1 m, broad spacing distributions, and only weak short-range clustering. Depth-resolved confocal analysis confirmed this sparse organization across larger biofilm volumes and revealed vertical stratification, with the highest bacterial volume fraction near the substrate and lower cell volume fraction toward the biofilm surface. Cryo-SEM further showed that mucoid biofilms contained aligned fibrillar structures within the biofilm interior, whereas PAO1 biofilms exhibited a denser, mesh-like matrix. These findings challenge prevailing views of biofilms as densely packed bacterial aggregates and establish a quantitative framework for understanding antimicrobial tolerance, cell-cell interactions, nutrient access, and biofilm mechanics in both clinically and environmentally relevant contexts.