BiofilmLightMap · Fluorescence-guided Correlative Imaging of Staphylococcal Biofilm Matrix Architecture

Staphylococcus aureus is a persistent global challenge in wound treatment and a leading cause of post-transplant complications. Its remarkable ability to evade therapy relies in part on the formation of biofilms—structured communities of bacterial cells embedded in an extracellular polymeric substances (EPS) matrix. While the composition of this matrix—extracellular DNA, polysaccharide inter-cellular adhesin (PIA), and secreted proteins—is qualitatively understood, little is known about the molecular structures adopted by these components during formation and maturation of biofilm. This gap hampers efforts to interpret biofilm physiology and design targeted anti-biofilm strategies. With current therapies relying on multi-drug antibiotic regimens administered over extended periods—often with limited success and frequent recurrence—there is an urgent need to re-conceptualize the biofilm as a tissue-like structure, whose protective and adaptive functions are governed by the EPS matrix.The proposed project aims to develop and apply a panel of fluorescent markers for the identification and spatial mapping of key EPS matrix components within native biofilms. These markers—targeting extracellular DNA and RNA (via metabolic labeling), amyloid-like proteins (FSB dye), membrane vesicles (fluorescent conjugates), and polysaccharides like PIA (lectin-based probes)—will be optimized for compatibility with both fluorescence microscopy and cryo-electron tomography (cryo-ET). Marker performance will be evaluated in Staphylococcus aureus and Staphylococcus epidermidis strains, selected for their distinct EPS compositions. Initial large-volume mapping will be performed using FIB-SEM tomography to classify architectural phenotypes and guide targeted lamella preparation for cryo-ET. By resolving the in situ organization of EPS matrix components, this project will provide structural insights into biofilm resilience and lay the groundwork for novel diagnostic and therapeutic strategies.