Towards chemical analysis of nanostructures in biofilms I: imaging of biological nanostructures.

Due to their direct influence on the stability of bacterial biofilms, a better insight into the nanoscopic spatial arrangement of the different extracellular polymeric substances (EPS), e.g., polysaccharides and proteins, is important for the improvement of biocides and for process optimization in wastewater treatment and biofiltration. Here, the first application of a combination of confocal laser-scanning microscopy (CLSM) and atomic force microscopy (AFM) to the investigation of river-water biofilms and related biopolymers is presented. AFM images collected at selected areas of CLS micrographs dramatically demonstrate the heterogeneity of biofilms at the nanometer scale and the need for a chemical imaging method with nanoscale resolution. The nanostructures (e.g., pili, flagella, hydrocolloids, and EPS) found in the extracellular matrix are classified according to shape and size, which is typically 50-150 nm in width and 1-10 nm in thickness, and sets the demands regarding spatial resolution of a potential chemical imaging method. Additionally, thin layers of the polysaccharide alginate were investigated. We demonstrate that calcium alginate is a good model for the EPS architecture at the nanometer scale, because of its similar network-like structure.