Framework Integration Engineered Asymmetric Cyanine Dyes for <i>In Vivo</i> High-Performance NIR-II Imaging and Multiplexed Labeling

The development of small-molecule near-infrared-II (NIR-II) fluorophores for high-contrast bioimaging is desirable, but challenging. Here, we report the design of a new class of NIR-II dyes, named <b>AFlavs</b>, created via framework integration of flavylium and polymethine scaffolds. These asymmetric molecules exhibit high brightness (227-2396 M^-1 cm^-1), superior photostability, and strong resistance to quenching, making them ideal for advanced bioimaging. Among them, <b>AFlav-4</b> stands out for its performance in vascular imaging, including tumor vasculature disruption, ischemia-reperfusion evaluation, and bone-targeted delivery via nanoparticles. Notably, <b>AFlavs</b> enabled two-, three-, and four-color multiplex imaging via more than 100 nm absorption separation, achieving spectral orthogonality uncommon among the currently reported small-molecule NIR-II fluorophores. These findings demonstrate the potential of <b>AFlav</b>s for high-resolution, multicolor, and long-term NIR-II imaging <i>in vivo</i>.