Deep-Penetrating and High-Resolution Continuous-Wave Nonlinear Microscopy Based on Homologous Dual-Emission Upconversion Adaptive Optics

Abstract

Lanthanide-doped upconversion nanoparticles (UCNPs) are emerging as innovative nonlinear probes in biomedical studies, offering the unique capability to simultaneously emit both visible (VIS) and near-infrared (NIR) photons under continuous-wave (CW) NIR excitation. However, deep-tissue high-resolution imaging remains challenging due to the trade-off between VIS emission (higher resolution, limited penetration) and NIR emission (deeper penetration, lower resolution). Here we present a CW nonlinear microscopy based on homologous dual-emission upconversion adaptive optics, leveraging Tm³⁺/Yb³⁺ co-doped UCNPs’ dual 455 nm/800 nm emission: the 800 nm emission for aberration measurement (guide-star) in deep tissues and the 455 nm emission for high-resolution imaging at matching depths. Using a home-built nonlinear laser scanning microscope with a 975 nm CW laser, we achieved near-diffraction-limited imaging (480 nm laterally) at a 500 μm depth in the mouse brain environment with significant optical aberrations. This strategy expands UCNPs’ applications and innovates the exploration of deep-tissue optical features.