808 nm-excited upconversion single-particle imaging for long-term tracking of cargo transport in live neurons

Upconversion nanoparticles (UCNPs) activated by 808 nm excitation hold promise for bioimaging due to minimized water absorption and tissue heating. However, their luminescence efficiency and single-particle performance in complex biological systems remain limited. Here, we present a rationally designed core-multishell UCNP structure (NaYbF 4 : x%Er@NaYF 4 : y%Yb@NaYF 4 : z%Nd, 10 %Yb@NaYF 4 ) optimized for enhanced upconversion luminescence (UCL) at the single-particle level under 808 nm excitation. By systematically tuning Er 3+ concentration in the emissive core, Yb 3+ in the energy migration shell, and Nd 3+ in the energy-harvesting layers, we achieved efficient energy spatial management. Single-particle analysis revealed power-dependent UCL saturation, with 12 % Er 3+ doping in the core, 10 % Yb 3+ doping in the migration shell, and 30 % Nd 3+ doping in the energy-harvesting layer yielding maximal brightness. These UCNPs were further surface-functionalized for live-cell imaging and enabled continuous cargo tracking in hippocampal neurons for over 30 min under 808 nm excitation. Trajectory analysis revealed hitchhiking-like transport dynamics within axons. This work highlights the potential of UCNPs for advanced biological imaging and mechanistic insights into nanoscale cargo dynamics in neurons. • Developed core-multishell upconversion nanoparticles (UCNPs) ,maximizing brightness under 808 nm excitation. • Achieved >30 min continuous cargo tracking in live hippocampal neurons using 808 nm excitation.∙Revealed hitchhiking-like axonal transport dynamics with minimal photodamage and heating effects. • Achieved >30 min continuous tracking of vesicular transport in live neurons using 808 nm light, minimizing photodamage and heating.