Real-Time Label-Free Monitoring of Nanoparticle–Cell Interactions in Single Tetrahymena thermophila by Mass Cytometry

Quantitative characterization of nanoparticle (NP)–cell interactions by aquatic organisms remains analytically challenging due to the rapid, heterogeneous, and dynamic nature of NP–cell processes. In this study, we presented a label-free, real-time analytical methodology that facilitated high-temporal-resolution analysis of NP–cell interactions in Tetrahymena thermophila by mass cytometry (CyTOF). By utilizing 88Sr as a cellular fingerprint marker of T. thermophila, we achieved subsecond detection resolution of NP–cell interaction signals under continuous monitoring conditions. The interaction kinetics of NPs, evaluated using I0/T1/2 were significantly dependent on temporal resolution, with values obtained at high temporal resolution being approximately 2-fold higher than those derived from low-resolution sampling. This strategy reduced information loss by 39% compared to long-interval sampling. Furthermore, a distinct subpopulation exhibiting high NPs-associated signals was identified, with its proportion increasing from 1.4 ± 0.3 to 9.3 ± 0.5% over 10 h, which provided a quantitative basis for targeted characterization of functionally distinct cellular states at the single-cell level. Overall, this method achieved continuous, high-resolution, and high-throughput quantification of NP–cell interaction dynamics, providing an analytical framework for investigating time-dependent NP behavior at the single-cell level.