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.