Upconversion nanoparticles assisted multi-photon fluorescence saturation microscopy

Bright and photo-stable luminescent nanoparticles held great potential for bioimaging, long-term molecular tracking. Rare-earth-doped upconversion nanoparticles (UCNPs) have been recently discovered with unique properties for Stimulated Emission Depletion (STED) super-resolution microscopy imaging. However, this system strictly requires optical alignment of concentric excitation and depletion beams, resulting in cost, stability, and complicity of the system. Taking the advantage of intermediate state saturation in UCNPs, emission saturation nanoscopy has been developed as a simplified modality by using a single doughnut excitation beam. In this work, we report that the emission saturation curve of fluorescence probes can modulate the performance of multi-photon emission saturation nanoscopy. With the precise synthesis of UCNPs, we demonstrate the resolution of this new imaging approach can be improved with five parameters, including emission band, activator doping, excitation power, sensitizer doping, core-shell. This approach opens a new strategy to a simple solution for super-resolution imaging and single molecule tracking at low cost, suggesting a large scope for materials science community to improve the performance of emission saturation nanoscopy.