In fiber suspensions with low optical contrast, the in situ characterization of structural properties with conventional microscopy methods fails. However, overlaying subsequent images of multiple particle tracking (MPT) videos including short trajectories usually discarded in MPT analysis allowed for direct visualization of individual fibers and the network structure of lyophilized collagen I (Coll) distributed in hydrochloric acid solutions. MPT yielded a broad distribution of mean square displacements (MSDs). Freely diffusing tracer particles yielded viscosities indicating that, irrespective of concentration, a constant amount of Coll is dissolved in the aqueous phase. Particles found elastically trapped within fibrous Coll structures exhibited a broad range of time-independent MSDs and we propose a structure comprising multiple fiber bundles with dense regions inaccessible to tracers and elastic regions of different stiffness in between. Bulky aggregates inaccessible to the 0.2 μm tracers exist even at low Coll concentrations, a network of slender fibers evolves above the sol–gel transition and these fibers densify with increasing Coll concentration. This novel MPT-based imaging technique possesses great potential to characterize the fiber distribution in and structural properties of a broad range of biological and technical suspensions showing low contrast when imaged with conventional techniques. Thus, MPT imaging and microrheology will help to better understand the effect of fiber distribution and network structure on the viscoelastic properties of complex suspensions
