Imaging of bacterial biofilms in solution by atmospheric scanning electron microscopy

Biofilm is one of the common morphological and ecological states of microbes, and forms on biotic and abiotic surfaces. Within a biofilm, microbes are embedded in a self-produced extracellular matrix (ECM) composed of proteins, polysaccharides and/or extracellular nucleic acids. To date, the high-resolution structural information required to understand the mechanisms leading to their formation and develop novel therapeutic strategies for biofilm-associated infections, is lacking. Here, we developed atmospheric scanning electron microscopy (ASEM) [1] to visualise biofilms immersed in aqueous solution, including biofilms formed by the Gram-positive coccus Staphylococcus aureus and the Gram-negative bacillus Escherichia coli [2]. Since ASEM allows a biofilm cultured on electron-transparent film windows to be observed by an inverted SEM from below, it was possible to study biofilm formations near the substrate and the ECM at high resolution. Membrane vesicle production within biofilms was imaged and shown to be linked to the trafficking of several cargos, including cytoplasmic proteins, periplasmic aggregated proteins, toxins, and RNA. In the ECM, proteins (e.g., the staphylococcal biofilm matrix protein Eap [3,4], staphylococcal protein A, delicate spiral flagella and straight curli fibrils) and filamentous extracellular DNA networks connecting bacterial cells were visualised. Collectively, our results suggest that ASEM, combined with the efficient and/or high-throughput labelling options presented, is a broadly applicable approach for microbial research and diagnostic purposes. © 2016, Oxford University Press. All rights reserved.