Single-Molecule\nSpectral Fluctuation Originates from\nthe Variation in Dipole Orientation Connected to Accessible Vibrational\nModes

Fluctuation in fluorescence emission of an immobilized\nsingle molecule\nis typically ascribed to the chromophore’s intrinsic structural\nconformations and the influence of local environmental factors. Despite\nextensive research since its initial observation, a direct connection\nbetween these spectral fluctuations and the rearrangement of emission\ndipole orientations has remained elusive. Here, we elucidate this\nfundamental molecular behavior and its underlying mechanisms by employing\nunique single-molecule multidimensional tracking to simultaneously\nmonitor both the emission spectrum and the three-dimensional dipole\norientation of individual fluorophores. We present compelling evidence\ndemonstrating a correlation between spectral fluctuations and dipolar\nrearrangements at room temperature. Our observations reveal that variations\nin the radiative relaxation probabilities among different vibronic\nemission bands, coupled with the interaction of associated vibrational\nmodes, drive these spectral fluctuations. We identify significant\nout-of-plane dipole reorientations during pronounced spectral fluctuations,\ncommonly known as spectral jumps, which primarily arise from transitions\nbetween dominant vibrational modes. Furthermore, we emphasize the\npotential for constructing vibrational spectra and optical nanoscopy\nwith vibrational specificity, leveraging the vibronic emissions from\nsingle emitters.