The pillars of (biofilm) creation: widefield deconvolution epifluorescence microscopy reveals a novel developmental model for filamentous fungal biofilms

Previous three-dimensional (3D) analyses of filamentous fungal biofilms have suggested an organized mycelial structure. By using Widefield Deconvolution Epifluorescence Microscopy (WDEM), we describe a novel structural model for fungal biofilms that could help explain important aspects of their biology, such as antimicrobial resistance and tolerance. WDEM has provided the most detailed and visually striking 3D images of fungal biofilm architecture to date, revealing highly organized mycelial development and distinct exopolysaccharide structures. A few hours after conidial germination, originating from the substrate-adherent mycelium, groups of hyphae converge to form numerous cylindrically arranged bundles that grow vertically. These hyphae secrete a substantial amount of exopolysaccharides that fuse them, creating a cohesive, hyperboloid-shaped chimney structure. These exopolysaccharide chimneys are numerous, their formation happens continuously throughout development, and they are distributed across the entire biofilm. As the biofilm matures, the openings of adjacent chimneys coalesce, isolating the vegetative mycelium growing between them from the external environment. We propose that this isolation establishes that exchanges with the exterior occur through the chimney flues, allowing them to become molecular filters and to regulate the inflow of xenobiotics. This highly organized architecture reveals a previously unknown level of structural complexity in sessile submerged fungal biofilms.