Nanoscale chemical imaging of Bacillus subtilis spores by combining tip-enhanced Raman scattering and advanced statistical tools.

Tip-enhanced Raman Scattering (TERS) has recently emerged as a powerful spectroscopic technique capable of providing subdiffraction morphological and chemical information on samples. In this work, we apply TERS spectroscopy for surface analysis of the Bacillus subtilis spore, a very attractive biosystem for a wide range of applications regulated by the spore surface properties. The observed 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 applications.