Low irradiance multiphoton imaging with alloyed lanthanide nanocrystals

Multiphoton imaging techniques that convert low-energy excitation to higher energy emission are widely used to improve signal over background, reduce scatter, and limit photodamage. Lanthanide-doped upconverting nanoparticles (UCNPs) are among the most efficient multiphoton probes, but even UCNPs with optimized lanthanide dopant levels require laser intensities that may be problematic. Here, we develop protein-sized, alloyed UCNPs (aUCNPs) that can be imaged individually at laser intensities >300-fold lower than needed for comparably sized doped UCNPs. Using single UCNP characterization and kinetic modeling, we find that addition of inert shells changes optimal lanthanide content from Yb(3+), Er(3+)-doped NaYF(4) nanocrystals to fully alloyed compositions. At high levels, emitter Er(3+) ions can adopt a second role to enhance aUCNP absorption cross-section by desaturating sensitizer Yb(3+) or by absorbing photons directly. Core/shell aUCNPs 12 nm in total diameter can be imaged through deep tissue in live mice using a laser intensity of 0.1 W cm(−2).