Temporal analysis of relative distances (TARDIS) is a robust, parameter-free alternative to single-particle tracking.

In single-particle tracking, individual particles are localized and tracked over time to probe their diffusion and molecular interactions. Temporal crossing of trajectories, blinking particles, and false-positive localizations present computational challenges that have remained difficult to overcome. Here we introduce a robust, parameter-free alternative to single-particle tracking: temporal analysis of relative distances (TARDIS). In TARDIS, an all-to-all distance analysis between localizations is performed with increasing temporal shifts. These pairwise distances represent either intraparticle distances originating from the same particle, or interparticle distances originating from unrelated particles, and are fitted analytically to obtain quantitative measures on particle dynamics. We showcase that TARDIS outperforms tracking algorithms, benchmarked on simulated and experimental data of varying complexity. We further show that TARDIS performs accurately in complex conditions characterized by high particle density, strong emitter blinking or false-positive localizations, and is in fact limited by the capabilities of localization algorithms. TARDIS' robustness enables fivefold shorter measurements without loss of information.