Single-Molecule Spectral Fluctuation Originates from the Variation in Dipole Orientation Connected to Accessible Vibrational Modes

Fluctuation in fluorescence emission of an immobilized
single molecule
is typically ascribed to the chromophore’s intrinsic structural
conformations and the influence of local environmental factors. Despite
extensive research since its initial observation, a direct connection
between these spectral fluctuations and the rearrangement of emission
dipole orientations has remained elusive. Here, we elucidate this
fundamental molecular behavior and its underlying mechanisms by employing
unique single-molecule multidimensional tracking to simultaneously
monitor both the emission spectrum and the three-dimensional dipole
orientation of individual fluorophores. 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 constructing vibrational spectra and optical nanoscopy
with vibrational specificity, leveraging the vibronic emissions from
single emitters.