  A quantitative single-molecule tip-enhanced Raman spectroscopy (TERS) study
at room temperature remained a challenge due to the rapid structural dynamics
of molecules exposed to air. Here, we demonstrate the single-molecule level
hyperspectral TERS imaging of brilliant cresyl blue (BCB) at room temperature
for the first time, along with quantitative spectral analyses. Freeze-frame
approach using a thin Al2O3 capping layer, which suppresses spectral diffusions
and inhibits chemical reactions and contaminations in air, enabled reliable and
robust chemical imaging. For the 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, we reveal the conformational heterogeneity
at the single-molecule level. This work provides a facile way to investigate
the single-molecule properties in interacting media, expanding the scope of
single-molecule vibrational spectroscopy.
