  Cryogenic tip-enhanced Raman spectroscopy (TERS) studies have revealed
single-molecule dynamics; however, understanding the nature of single molecules
in ambient conditions has remained challenging. Here, we demonstrate the
hyperspectral TERS imaging of single brilliant cresyl blue (BCB) molecules,
along with quantitative spectral analyses, revealing their conformational
heterogeneity in ambient conditions. Robust single-molecule imaging is enabled
by encapsulating the molecules with a thin Al$_{2}$O$_{3}$ film, which
suppresses spectral diffusions and inhibits chemical reactions and
contaminations in air. For the single molecules resolved spatially in the TERS
image, a clear Raman peak variation up to ~7.5 cm$^{-1}$ is observed, which
cannot be found in molecular ensembles. From density functional theory-based
quantitative analyses of the varied TERS peaks of single molecules, we reveal
the single-molecule conformational heterogeneity at room temperature. This work
provides a facile way to investigate the single-molecule properties in
interacting media, expanding the scope of single-molecule vibrational
spectroscopy studies.
