Photophysical lock-in detection enables background-free upconversion emission imaging

Lanthanide-based upconversion nanoparticles (UCNPs) have attracted considerable attention in biomedical applications, due to their anti-Stokes shifted emission enabling autofluorescence-free signal detection. However, residual excitation light can still interfere with their relatively weak emission signals. 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 propose a photophysical lock-in detection (PP-LID) approach based on the discovery that UCNPs can act as frequency mixers in response to intensity-modulated excitation. Particularly, excitation modulated at multiple base frequencies can generate additional spectral components at the beat frequencies (BFs) between the base modulation frequencies. These signals are resolvable by frame-rate-limited cameras, devoid of ambient and residual excitation light, and can be adapted through nanoparticle engineering. Extracting BF signals by PP-LID thus provides a strategy to significantly enhance signal-to-background conditions in UCNP-based bioimaging and biosensing.