Low irradiance multiphoton imaging with alloyed lanthanide nanocrystals

Bining Tian, Angel Fernandez-Bravo, Hossein Najafiaghdam, Nicole A. Torquato, M. Virginia P. Altoe, Ayelet Teitelboim, Cheryl A. Tajon, Yue Tian, Nicholas J. Borys, Edward S. Barnard, Mekhail Anwar, Emory M. Chan, P. James Schuck, Bruce E. Cohen

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 Yb3+, Er3+-doped NaYF4 nanocrystals to fully alloyed compositions. At high levels, emitter Er3+ ions can adopt a second role to enhance aUCNP absorption cross-section by desaturating sensitizer Yb3+ 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.