- Biological systems can be highly heterogeneous at the nanometer scale. AFM-based setups for tip-enhanced Raman spectroscopy (TERS) allow the combination of an analytical standard tool for biological samples (confocal laser-scanning microscopy, CLSM) with an imaging technique that provides nanometer-scale resolution (atomic force microscopy, AFM). Additionally, Raman microscopy and TERS provide chemical information of complex biological systems at the sub-micrometer scale. The arrangement of the different EPS in the biofilm matrix as well as their interactions with each other and with the cell surfaces are 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. atomic force microscopy (AFM)). Nanoscale Imaging Imaging of selected parts of a CLS micrograph by AFM provides detailed information on nanometer-sized structures as shown in Fig. 1 for a bacterial biofilm sample.
The spatial resolution of the AFM enables imaging of nanometer-sized extracellular structures in biofilms, such as extracellular polymeric substances (EPS), alginate bundles, flagella, pili, and hydrocolloids (see Fig. 2). Knowledge about their distribution has implications in many fields where biofilm formation plays a role, such as medicine, industrial processes, wastewater treatment and the environment.
Nanoscale Chemical Analysis For label-free chemical characterization of biological samples with nanometer-scale spatial resolution, we demonstrated the feasibility of TERS , which provides fingerprint spectra reflecting the molecular composition of a sample area with a diameter of down to 20-50 nm. Fig. 3 shows the TERS analysis of nanometer-sized calcium alginate fiber bundles, a stabilizing agent that can be found in bacterial biofilms.
Applications Biological applications of our analytical tools for the nanoworld include: - Bacterial biofilms The goal of further studies is to improve TERS towards 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. TERS can then be applied in many other fields of medicine and biology.
Dr. Thomas Schmid, schmid@org.chem.ethz.ch Literature:
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