KCS

Single-molecule TERS experiment in ambient conditions is of fundamental significance because the molecular properties can change depending on temperature and atmosphere. Specifically, understanding the conformational heterogeneity of single molecules in the non-equilibrium state is highly desirable because it can address many fundamental questions regarding structure and function in many biological systems, such as protein folding and RNA dynamics. Yet, understanding the single-molecule conformational heterogeneity in ambient conditions has remained challenging due to the rapid structural dynamics and spectral diffusions. We have performed robust hyperspectral TERS imaging of single brilliant cresyl blue (BCB) molecules in ambient conditions through the novel freeze-frame approach using a thin Al₂O₃ capping layer to suppress the spectral diffusions of single isolated molecules. Through quantitative spectral analyses of the measured TERS spectra, we reveal the conformational heterogeneity of single molecules. We believe this work provides an optimal protocol to investigate the single-molecule properties in interacting media, expanding the scope of single- molecule vibrational spectroscopy studies.