Characterizing Rotational Dynamics and Heterogeneity via Single-Molecule Intensity Measurements.

Dynamic heterogeneity in glassy systems has typically been characterized at the single-molecule level by extracting rotational relaxation time scales from linear dichroism (LD) collected via two orthogonally polarized channels. However, in such measurements, localization precision is diminished due to photons lost relative to collection in a single detection channel. This poses challenges in characterizing rotational and translational dynamics simultaneously, as translational measurements require high localization precision. In this paper, we present a method for extracting rotational dynamics of glassy systems at the single-molecule level from intensity fluctuations of fluorescent probe molecules in a wide-field configuration without the use of a polarizing optical component. Through numerical analysis, we show that LD and intensity measurements probing rotational dynamics report similar, approximately second-order rotational correlation decays even at low signal to noise. Thus, within the assumptions of small, isotropic rotations, LD and intensity autocorrelation analysis should provide identical information on the time scale and heterogeneity of rotational dynamics. We then present experimental results that validate this numerical result, with direct comparison of LD and intensity-based approaches across probe molecules in both polymeric and small-molecule glass formers as well as across optical configurations. Our results demonstrate moderate correlation on a per-probe basis between rotational time scales obtained from both approaches, with deviations consistent with those expected in a dynamically heterogeneous system. We envision that this easily accessible strategy will be of use across disciplines for characterizing single-molecule rotational dynamics in limited signal situations and/or when high-precision localization is required.