Targeted single-particle tracking with upconverting nanoparticles

Abstract

Single-particle tracking (SPT) is a powerful technique to elucidate the behavior of biomolecules inside or on the surface of living cells in real time. SPT is based on labeling the component of interest with a phosphor or a light-scattering gold nanoparticle, and visualizing its motion by real time microscopy. However, SPT generally suffers from suboptimal performance of phosphors. For example, fluorescent dyes have poor photostability, while quantum dots suffer from sporadic blinking that hampers track acquisition and interpretation. Light-scattering gold nanoparticles require custom experimental setups and relatively large particle size for tracking purposes. In recent years, upconverting nanoparticles (UCNPs) have emerged as a promising phosphor that enables backgroundless anti-Stokes luminescence imaging. They are also attractive for SPT applications due to their extreme photostability and non-blinking luminescence. Previously, SPT has been used to monitor UCNP endocytosis in cells. However, targeted SPT with UCNPs that specifically bind to biomolecules of interest has not yet been achieved. In this work, we provided a first proof-of-concept experiment of targeted SPT with UCNPs and showed the superior performance of UCNPs compared to organic dyes and quantum dots. As a benchmark system to assess the performance of UCNPs, we tracked FcεRI receptors on the surface of RBL-2H3 cell membranes using UCNP-IgE conjugates and obtained good agreement with data from the literature. Importantly, the versatility of the biotin-streptavidin architecture of our system allows UCNPs to be used to track a wide range of targets. Overall, this report lays the foundation for UCNP applications in targeted SPT.