Ratiometric fluorescent probes in the second near-infrared window (NIR-II) with a self-calibration function are sought-after for reliable imaging of physiological and pathological processes. Nevertheless, current ratiometric NIR-II fluorescent probes usually show severe spectral overlap in the emission channel, resulting in inevitable sacrifice of the emission intensity of the probe and compromised imaging quality in the NIR-II region. To address these challenges, we developed a novel dual-excitation ratiometric NIR-II fluorescence nanoplatform (DERF-NP), in which the intensity ratio of the same full-wavelength emission from 1000 to 1700 nm under two non-overlapping monochrome excitations with distinct responses is conventionally defined as the quantification parameter. As a proof of concept, DERF-NO, our test case of a ratiometric NIR-II nanoprobe for NO imaging with high sensitivity and quality was developed based on the energy transfer strategy, which showed increased emission with excitation at 660 nm and constant emission with excitation at 808 nm upon activation of NO. In vivo ratiometric NIR-II fluorescence imaging with DERF-NO successfully tracked macrophage polarization and lymphatic metastasis, suggesting the extensive distribution and critical role of macrophages in tumorigenesis and progression. This dual-excitation ratiometric imaging strategy may provide a novel approach for designing ratiometric NIR-II fluorescent probes, and has great application potential for intravital imaging analysis.
