Near-infrared (NIR) fluorescent probes are among the most attractive chemical tools for biomedical imaging. However, their in vivo applications are hindered by albumin binding, generating unspecific fluorescence that masks the specific signal from the analyte. Here, combining experimental and docking methods, we elucidate that the reason for this problem is an acceptor (A) group-mediated capture of the dyes into hydrophobic pockets of albumin. This pocket-capturing phenomenon commonly applies to dyes designed under the twisted intramolecular charge-transfer (TICT) principle and, therefore, represents a generic but previously unidentified backdoor problem. Accordingly, we create a new A group that avoids being trapped into the albumin pockets (pocket-escaping) and thereby construct a NIR probe, BNLBN, which effectively prevents this backdoor problem with increased imaging accuracy for liver fibrosis in vivo. Overall, our study explains and overcomes a fundamental problem for the in vivo application of a broad class of bioimaging tools.

近红外 (NIR) 荧光探针是生物医学成像中最具吸引力的化学工具之一。然而，它们的体内应用受到白蛋白结合的阻碍，产生非特异性荧光，掩盖了来自分析物的特异性信号。在这里，结合实验和对接方法，我们阐明了这个问题的原因是受体 (A) 基团介导的染料捕获到白蛋白的疏水口袋中。这种口袋捕获现象通常适用于根据扭曲分子内电荷转移 (TICT) 原理设计的染料，因此代表了一个普遍但先前未识别的后门问题。因此，我们创建了一个新的 A 基团，可以避免被困在白蛋白口袋中（口袋逃逸），从而构建 NIR 探针 BNLBN，它可以有效防止这种后门问题，提高体内肝纤维化的成像精度。总的来说，我们的研究解释并克服了广泛的生物成像工具在体内应用的一个基本问题。