Long-Term Single-Particle Tracking of Membrane Proteins Enabled by Upconversion Alkaline-Earth Nanoprobes.

Abstract

Single-particle tracking (SPT) provides mechanistic insights into individual biomolecules but suffers from the limited photostability of conventional probes. Upconversion SPT (uSPT), employing lanthanide-doped nanoparticles (UCNPs) as probes, enables extended imaging durations; however, its broader application is hindered by the trade-off between nanoparticle size and brightness. To overcome this limitation, we designed ∼10 nm core-shell-shell UCNPs incorporating strontium-based alkaline-earth materials, achieving both compact dimensions and enhanced luminescence. These engineered UCNPs exhibit a 4.6-fold increase in brightness compared to that of conventional sodium-based counterparts. Using these probes, we tracked epidermal growth factor receptor (EGFR) dynamics and identified four distinct motion states (immobile, confined, free, and directed) highlighting the motional heterogeneity and reversible transitions of individual EGFR. Treatment with latrunculin A markedly altered these motional states, underscoring the critical role of the cytoskeleton in regulating EGFR dynamics. Collectively, our findings demonstrate the utility of uSPT for probing membrane protein behavior and dissecting complex cellular processes.