Multiplexed Single‐Particle Imaging Enabled by Modulation of Er ³⁺ Energy‐Level Populations

Abstract

Abstract Single‐particle tracking (SPT) offers critical insights into nanoscale molecular dynamics, but is limited by short tracking durations due to irreversible photobleaching and the technical complexity of multicolor imaging. Here, a non‐photobleaching ratiometric imaging strategy is developed based on lanthanide‐doped upconversion nanoparticles (UCNPs), exploiting their intrinsic, tunable red‐to‐green (R/G) emission ratios for multiplexed SPT. Single‐particle characterization reveals over 10‐fold tunability. Mechanistic investigations show that this ratiometric behavior is governed by Yb 3+ excitation density, which modulates the energy distribution within energy levels of Er 3+ ions. Specifically, high Yb 3+ excitation densities enhance three‐photon transitions, favoring red emission, while lower densities promote two‐photon upconversion and green emission. Both processes proceed through a shared energy level of 2 H 11/2 , leading to competitive energy transfer dynamics. Based on this competition mechanism, a quantitative relationship is further established between UCNPs structure and the resulting R/G emission ratio, allowing reliable prediction of spectral output across different designs. Leveraging this tunable ratiometric principle, simultaneous five‐color single‐particle imaging is demonstrated with a misidentification rate below 5%. This strategy is further applied to visualize receptor‐mediated endocytosis in live cells. This work highlights the advantages of upconversion luminescence‐based R/G ratio discrimination for long‐term, multicolor SPT, offering a simple and reliable tool for probing complex biological processes.