Tracer transport provides excellent insight to investigate the local environments of inhomogeneous systems in n cellular media as well as gel and polymer networks (see Fig. 1a for instance). However, the transient immobility during the transport is obscured by the localization error (LE), the uncertainty to locate the exact position in super-localization. Because the dimension of localization error (~30-50 nm) is an order of magnitude larger than a molecular probe (~1-2 nm). Therefore, the super-localized SMT cannot discriminate between the momentary stop and the localized motion within the  (Fig. 1b). We propose a concept that authentic pauses within the can be revealed by probing the hindrance of SM reorientational dynamics since the translation of passive tracers in the condensed phase is associated with fast dipolar rotation. Therefore, identifying instances where a tracer is rotationally immobile would serve as a clear indicator for its stasis; conversely, the fast dipolar reorientation implies localized motion within  (Fig. 1c). Here, we show how polarized emission intensities of mobile tracers at two orthogonal channel reveals the tumbling propensity of SMs during random walks (Fig. 1d).

Polarization-resolved single-molecule tracking (PR-SMT) has been developed to retrieve emission anisotropy (LD) at each super-localized position which provides a detailed description of tracer transport in dense media (Fig. 1e, f). As a case study, I investigated the transport of single rhodamine 6G (Rh6G) molecules above the glass-transition temperature of plasticized polyvinylpyrrolidone (PVP) film. To obtain quantitative information on the occurrence of genuine stops and locally mobile events, I developed an algorithm to analyze the PR-SMT trajectory (Fig. 1g, h). I have selected 100 SMs for statistical assessment and identified the time-points of hindered or facile dipolar reorientation in each SM trajectory within the LE. The fraction of locally mobile and genuinely static events are compared with the traditional SMT method as a function of the dynamic range in each trajectory.

My analyses reveal that except few SMs, the vast majority of tracers are static for almost entire trajectory durations at lower dynamic range; however, tracers within the LE are quite diverse at elevated dynamic range reflecting a lesser duration of genuine stops compared to the conventional SMT. My PR-SMT data analyses on 100 Rh6G molecules reveal the frequent occurrence of authentic pauses during tracer navigation, demonstrating the intermittently translational and rotational immobilization for most probes (Fig. 1g, h).  Such results indicate that Rh6G molecules serendipitously encounter rigid polymer cavities during the transport, which renders the complete immobilization of the probe molecule. The finding implies the existence of nanoscale glass-like domains sparsely distributed in a predominantly deep-rubbery polymer network far above the glass transition.