Biological samples can be highly heterogeneous on the nanometer scale. Bacterial adhesion and biofilm formation, for example, are controlled by nanometer-sized structures of different chemical composition, such as pili, flagella, and extracellular polymeric substances (EPS). Biofilms are the predominant life form of bacteria in nature, which can occur in technical systems as well (e.g. biofouling in industrial plants, wastewater treatment). The arrangement of the different EPS (e.g. polysaccharides, proteins, humic substances) in the biofilm matrix is largely unknown, because microscopy techniques used so far were restricted in spatial resolution to the micrometer and higher nanometer range (e.g. confocal laser-scanning microscopy, CLSM) or did not provide any chemical information (e.g. AFM). Due to their direct influence on the stability of biofilms, a better insight into the distribution of the different extracellular nanostructures (e.g. pili, flagella, hydrocolloids, EPS) is important for the improvement of biocides and for process optimization in wastewater treatment.
Recently, the first application of a combined CLSM-AFM method to study and analyze biofilms was presented [1]. It allows AFM imaging of selected areas of a CLS micrograph with nanometer-scale resolution revealing bacteria, pili, flagella, and EPS. For nanometer-scale chemical analysis, the feasibility of tip-enhanced Raman spectroscopy (TERS) was demonstrated, where Ag-coated AFM tips [2] in the laser focus enhance the Raman signal, yielding chemical / spectroscopic information with a lateral resolution of down to 20–50 nm. Alginates in the form of nanometer-sized fibers and hydrogels were used as model samples for biofilms and investigated successfully by AFM and TERS.
The goal of further studies is to improve TERS to become a robust tool for the analysis of biological samples, which allows, for example, the elucidation of the distribution of different biopolymers (e.g. polysaccharides and proteins) inside the biofilm matrix and can be applied in many other fields of medicine and biology.
[1] T. Schmid, J. Burkhard, B.S. Yeo, A. Messmer, W. Zhang, R. Zenobi (2007) Anal. Chem., submitted.
[2] B.S. Yeo, T. Schmid, W. Zhang, R. Zenobi (2007) Anal. Bioanal. Chem., accepted. |