Fluorescence imaging represents a vital tool in modern biology, oncology and biomedical applications. Afterglow luminescence (AGL), which circumvents the light scattering and tissue autofluorescence interference associated with real-time excitation source, shows remarkably increased imaging sensitivity and depth. Here we present a protocol for the design and synthesis of AGL nanoprobes with an aggregation-induced emission (AIE) effect to simultaneously red shift and amplify the afterglow signal for tumor imaging and image-guided tumor resection. The nanoprobe (AGL AIE dot) is composed of an enol ether format of Schaap’s agent and a near-infrared AIE fluorogen (AIEgen) (tetraphenylethylene-phenyl-dicyanomethylene-4H-chromene, TPE-Ph-DCM) to suppress the nonradiative dissipation pathway. Pre-irradiating AGL AIE dots with white light could generate singlet oxygen to convert Schaap’s agent to its 1,2-dioxetane format, thus initializing the AGL process. With the aid of AIEgen, the AGL shows simultaneously red shifted emission maximum (from ~540 nm to ~625 nm) and enhanced intensity (by 3.2-fold), facilitating better signal-to-background ratio, imaging sensitivity and depth. Intriguingly, the activated AGL can last for over 10 days. Compared with conventional approaches, our method provides a new solution to concurrently red shift and amplify afterglow signals for better in vivo imaging outcomes. The preparation of AGL AIE dots takes ~2 days, the in vitro characterization takes ~10 days (less than 1 day if not involving afterglow kinetic profile study) and the tumor imaging and image-guided tumor resection takes ~7 days. These procedures can be easily reproduced and amended after standard laboratory training in chemical synthesis and animal handling.

荧光成像是现代生物学、肿瘤学和生物医学应用中的重要工具。余辉发光 (AGL) 避免了与实时激发源相关的光散射和组织自发荧光干扰，显示出显着提高的成像灵敏度和深度。在这里，我们提出了一种设计和合成具有聚集诱导发射（AIE）效应的 AGL 纳米探针的协议，可同时红移和放大余辉信号，用于肿瘤成像和图像引导肿瘤切除。纳米探针（AGL AIE点）由Schaap试剂的烯醇醚形式和近红外AIE荧光剂（AIEgen）（四苯乙烯-苯基-二氰亚甲基-4H-色烯，TPE-Ph-DCM）组成，以抑制非辐射耗散途径。用白光预照射 AGL AIE 点可以产生单线态氧，将 Schaap 试剂转化为其 1,2-二氧杂环丁烷形式，从而初始化 AGL 过程。在 AIEgen 的帮助下，AGL 同时显示发射最大值红移（从 ~540 nm 到 ~625 nm）和增强的强度（3.2 倍），从而促进更好的信号背景比、成像灵敏度和深度。有趣的是，激活的AGL可以持续10天以上。与传统方法相比，我们的方法提供了一种新的解决方案，可以同时进行红移和放大余辉信号，以获得更好的体内成像结果。 AGL AIE 点的制备需要约 2 天，体外表征需要约 10 天（如果不涉及余辉动力学曲线研究，则不到 1 天），肿瘤成像和图像引导肿瘤切除需要约 7 天。经过化学合成和动物处理方面的标准实验室培训后，这些程序可以轻松复制和修改。