Correlative TERS imaging of B. subtilis spores

Tip-enhanced Raman Scattering (TERS) has recently emerged as a unique and powerful analytical tool to directly localize and identify proteins and their conformation in a complex environment at the nanoscale. This analytical technique relies on the combination of near-field scanning probe microscopy with plasmonic-enhanced Raman spectroscopy. In TERS, in fact, a metalled tip is used as a sort of optical nano-antenna that gives place to the a plasmonic amplification of the Raman signal only at the tip apex of the scanning probe, which, in turn, allows near field, sub-diffraction spatial resolution of the acquired spectral information. Moreover, TERS allows obtaining simultaneously topographic and chemical information of the analysed surface region, which constitutes an important step for surface analysis in its broadest sense. In this work, we apply TERS spectroscopy for surface analysis of the Bacillus subtilis spore, a very attractive bio-system for a wide range of applications regulated by the spore surface properties. The observed TERS spectra reflect the complex and heterogeneous environment explored by the plasmonic tip, therefore exhibiting significant point-to-point variations at the nanoscale. Herein, we demonstrate that TERS data processing via principal component analysis allows handling such spectral changes, thus enabling an unbiased correlative imaging based on TERS. Our experimental outcomes suggest a denser arrangement of both proteins and carbohydrates on specific spore surface regions simultaneously revealed by AFM phase imaging. Successful TERS analysis of spores' surface is useful for bacterial surface-display systems and drug-delivery.