Multiphoton imaging techniques that convert near infrared excitation to higher energy visible emission are widely used in complex systems to improve signal over background, reduce scatter, and limit photodamage. Multiphoton imaging relies on luminescent probes able to efficiently sum the energies of multiple incident photons, as well as lasers powerful enough to generate necessarily high fluences. Lanthanide-doped upconverting nanoparticles (UCNPs) have proven to be among the most efficient multiphoton probes, and single UCNPs can be imaged as laser intensities 6 orders of magnitude lower than the best 2-photon fluorophores. But even optimized lanthanide compositions still require laser intensities (&gt;105 W/cm2) that may be problematic for living systems. Here we use single UCNP characterization and kinetic models of lanthanide energy transfer to develop novel compositions that can be imaged at the single molecule level at fluences 200-fold lower than the most efficient previous compositions, and less than typical 1-photon confocal fluences. We find that in Yb- and Er-doped NaYF4 core/shell UCNPs, increasing activator and emitter concentrations both lead to significant drops in the excitation intensity required to image single nanocrystals, so that optimized UCNPs require just 500 W/cm2 to be imaged above background. These lanthanide-doped nanoparticles open up a new approach to bioimaging, using near infrared laser intensities far below those required for 2-photon fluorescence imaging by almost 10 orders of magnitude.

Lanthanide-doped nanocrystals engineered for low fluence multiphoton imaging

253rd National Meeting (2017)

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