Enhanced Single-Particle Upconversion Imaging via Energy Migration Boosting.

Lanthanide-doped upconversion nanoparticles (UCNPs) are promising bioimaging probes due to their exceptional photostability and minimal background interference. However, their limited single-particle brightness has hindered broader applications. The study addresses this challenge by enhancing energy migration (EM) between sensitizer Yb3+ to improve energy transfer efficiency to emitter Er3+. Nanoparticles are designed with a sensitizer/emitter-segregated core-shell-shell architecture (NaLu0.9Er0.1F4@NaYbF4@NaLuF4) to inhibit back energy transfer (BET) and then increased Yb3+ doping levels (NaLu0.9-xYbxEr0.1F4@NaYbF4@NaLuF4) to enhance EM into the core. UCNPs with an alloy-core of NaYb0.9Er0.1F4 exhibited the brightest upconversion luminescence, achieving over a tenfold enhancement compared to NaLu0.9Er0.1F4-core counterparts, highlighting the importance of EM. Further optimization of the Yb3+/Er3+ ratio and inert shell thickness (NaLuF4) maximized single-particle brightness. These optimized UCNPs enabled long-term tracking of axonal transport in live dorsal root ganglion (DRG) neurons. Using a Bayesian Hidden Markov Model, it quantitatively characterized resolved heterogeneous motion states and annotated trajectories with local spatiotemporal dynamics of retrograde, anterograde, and diffusive motions. The analysis revealed a kinesin-dynein coordination mechanism, where anterograde motion facilitates retrograde activation. It also examined the effects of inhibitors and stimulants on transport behavior. These findings establish upconversion single-particle tracking (uSPT) as a powerful tool for long-term, real-time monitoring of neuronal activities.