Polarization-resolved single-molecule tracking reveals strange dynamics of individual fluorescent tracers through a plasticized (rubbery) polymer network

Tracking the movement of fluorescent single-molecule (SM) tracers has provided several new insights on the local structure and dynamics in complex environments such as soft materials and biological systems. However, SM tracking (SMT) remains unreliable at molecular length scales, as the localization-error (LE) of SM trajectories (~30-50 nm) is considerably larger than size of molecular tracers (~1-3 nm). Thus, instances of tracer (im)mobility in heterogeneous media, which provide indicator for underlying anomalous-transport mechanisms, remains obscured within the realms of SMT. Since translation of passive tracers in an isotropic network is associated with fast dipolar rotation, we propose authentic pauses within LE can be revealed upon probing SM reorientational dynamics. Here, we demonstrate how polarization-resolved SMT (PR-SMT) can provide emission-anisotropy at each super-localized position, thereby revealing tumbling propensity of SMs during random walk. For Rhodamine 6G tracers undergoing heterogeneous transport in a hydrated polyvinylpyrrolidone (PVP) network, analyses of PR-SMT trajectories enabled us to discern instances of genuine immobility and localized motion within the LE. Our investigations on 100 SMs in hydrated (plasticized) PVP films reveal a wide distribution of dwell-times and pause-frequencies, which demonstrate that majority of probes intermittently experience complete translational and rotational immobilization. This indicates tracers serendipitously encounter compact, rigid polymer cavities during transport, implying the existence of nanoscale glass-like domains sparsely distributed in a redominantly deep-rubbery polymer network far above the glass transition. PR-SMT is simple to implement and opens up alternate avenues to interrogate transient (bio)molecular interactions leading to anomalous transport in inhomogeneous media.