Multicolor Long-Term Single-Particle Tracking Using 10 nm Upconverting Nanoparticles

Single-particle tracking (SPT) is a powerful technique to unveil molecular behaviors crucial to the understanding of many biological processes, but it is limited by factors such as probe photostability and spectral orthogonality. To overcome these limitations, we develop upconverting nanoparticles (UCNPs), which are photostable over several hours at the single-particle level, enabling long-term multicolor SPT. We investigate the brightness of core–shell UCNPs as a function of inert shell thickness to minimize particle size while maintaining sufficient signal for SPT. We explore different rare-earth dopants to optimize for the brightest probes and find that UCNPs doped with 2% Tm³⁺/30% Yb³⁺, 10% Er³⁺/90% Yb³⁺, and 15% Tm³⁺/85% Yb³⁺ represent the optimal probes for blue, green, and near-infrared emission, respectively. The multiplexed 10 nm probes enable three-color single-particle tracking on live HeLa cells for tens of minutes using a single, near-infrared excitation source. These photostable and multiplexed probes open new avenues for numerous biological applications.