Real-Time Label-Free Monitoring of Nanoparticle–Cell Interactions in Single <i>Tetrahymena thermophila</i> 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.