Lanthanide upconversion nonlinearity: a key probe feature for background-free deep-tissue imaging

Lanthanide-based upconversion nanoparticles (UCNPs) have attracted considerable attention in biomedical applications, largely due to their anti-Stokes shifted emission enabling autofluorescence-free signal detection. However, residual excitation light can still interfere with their relatively low brightness. While commonly used lock-in detection can distinguish weak signals from substantial random background, concurrently modulated residual excitation light is not eliminated. This remains a challenge, particularly under demanding experimental conditions. Here, we explore the inherent nonlinear response of UCNPs and discover that UCNPs can act as frequency mixers in response to intensity-modulated excitation. Particularly, modulated excitation with more than one base modulation frequency can generate additional low-frequency beating-signals. We show how these signals are resolvable by low-speed detectors such as cameras, are devoid of ambient and residual excitation light, and how they can be enhanced through nanoparticle engineering. Detection of beating-signals thus provides a strategy to significantly enhance signal-to-background conditions in UCNP-based bioimaging and biosensing.