Super‐Resolving Particle Diffusion Heterogeneity in Porous Hydrogels via High‐Speed 3D Active‐Feedback Single‐Particle Tracking Microscopy

Nanoparticle diffusion in 3D porous structures is critical to understanding natural and synthetic systems but remains underexplored due to limitations in traditional microscopy methods. Here, 3D Single-Molecule Active-feedback Real-time Tracking (3D-SMART) microscopy is used to resolve nanoparticle dynamics in agarose gels with unprecedented spatiotemporal resolution. This highlights "hopping diffusion", where particles intermittently escape confinement pockets, providing insights into hydrogel microstructure. Long, highly sampled trajectories enable the extraction of kinetic parameters, confinement sizes, and thermodynamic barriers. This study demonstrates 3D-SMART's ability to probe particle-environment interactions at super-resolution (≈10 nm in XY and ≈30 nm in Z) in 3D, offering new perspectives on nanoparticle diffusion and the structural dynamics of porous materials, with implications for drug delivery, material science, and biological systems.