Tip-enhanced Raman spectroscopy and related techniques in studies of biological materials

Biological materials can be highly heterogeneous at the nanometer scale. The investigation of nanostructures is often hampered by the low spatial resolution (e.g. spectroscopic techniques) or very little chemical information (e.g. atomic force microscopy (AFM), scanning tunneling microscopy (STM)) provided by analytical techniques. Our research focuses on combined instruments, which allow the analysis of the exactly same area of a sample by complementary techniques, such as AFM and Raman spectroscopy. Tip-enhanced Raman spectroscopy (TERS) combines the high spatial resolution of AFM or STM with the chemical information provided by Raman spectroscopy. The technique is based on enhancement effects known from surface-enhanced Raman scattering (SERS). In TERS the enhancing metallic nanostructure is brought to the sample by an AFM or STM tip. With a TERS-active tip, enhanced Raman signals can be generated from a sample area as small as 10-50 nm in diameter. AFM analysis of bacterial biofilms has demonstrated their heterogeneity at the nanometer scale, revealing a variety of nanostructures such as pili, flagella, and extracelullar polymers. TERS measurements of the biopolymers alginate and cytochrome c have yielded spectroscopic fingerprints even of such weak Raman scatterers, which in future can allow their localization in complex matrices. Furthermore, biofilms of the bacterium Halomonas meridiana were studied, which was found to be involved in the generation of the mineral dolomite. Only combined AFM–Raman analysis was able to identify the nanoglobules found in laboratory cultures of H. meridiana as dolomite nanoparticles. Our combined setups are and will be applied to the investigation of biofilms, fish spermatozoa as well as biological membranes.