Molecular-Based FRET Nanosensor with Dynamic Ratiometric NIR-IIb Fluorescence for Real-Time <i>In Vivo</i> Imaging and Sensing.

Abstract

Real-time fluorescence sensing can provide insight into biodynamics. However, few fluorescent tools are available to overcome the tissue scattering and autofluorescence interference for high-contrast <i>in vivo</i> sensing with high spatiotemporal resolution. Here, we develop a molecular-based FRET nanosensor (MFN) capable of producing a dynamic ratiometric NIR-IIb (1500-1700 nm) fluorescence signal under a frequency-modulated dual-wavelength excitation bioimaging system. The MFN provides reliable signals in highly scattering tissues and enables <i>in vivo</i> real-time imaging at micrometer-scale spatial resolution and millisecond-scale temporal resolution. As a proof of concept, a physiological pH-responsive nanosensor (MFN_pH) was designed as a nanoreporter for intravital real-time monitoring of the endocytosis dynamics of nanoparticles in the tumor microenvironment. We also show that MFN_pH allows the accurate quantification of pH changes in a solid tumor through video-rate ratiometric imaging. Our study offers a powerful approach for noninvasive imaging and sensing of biodynamics with micrometer-scale spatial resolution and millisecond-scale temporal resolution.