  Fluctuation in fluorescence emission of immobilized single molecule is
typically ascribed to the chromophore's intrinsic structural conformations and
the influence of local environmental factors. Despite extensive research over
several decades since its initial observation, a direct connection between
these spectral fluctuations and the rearrangement of emission dipole
orientations has remained elusive. In this study, we elucidate this fundamental
molecular behavior and its underlying mechanisms by employing unique
single-molecule multi-dimensional tracking to simultaneously monitor both the
emission spectrum and the three-dimensional dipole orientation of individual
fluorophore. For the first time, we present compelling evidence demonstrating a
correlation between spectral fluctuations and dipolar rearrangements at room
temperature. Our observations reveal that variations in the radiative
relaxation probabilities among different vibronic emission bands, coupled with
the interaction of associated vibrational modes, drive these spectral
fluctuations. We identify significant out-of-plane dipole reorientations during
pronounced spectral fluctuations - commonly known as spectral jumps - which
primarily arise from transitions between dominant vibrational modes.
Furthermore, we emphasize the potential for con-structing vibrational spectra
and optical nanoscopy with vibrational specificity, leveraging the vibronic
emissions from single emitters.
