Noninvasive and specific visualization of lysosomes by fluorescence technology is critical for studying lysosomal trafficking in health and disease and for evaluating new cancer therapeutics that target tumor cell lysosomes. To date, there are two basic types of lysosomal probes whose lysosomal localization correlates with lysosomal acidity and endocytosis pathway, respectively. However, the former may suffer from pH-sensitive lysosomal localization and alkalization-induced lysosomal enzyme inactivation, and the latter need long incubation time to penetrate cell membrane due to the energy-dependency of endocytosis process. In this work, a new class of two-photon fluorescent dyes, termed amino-Si-rhodamines (ASiRs), were developed, which possess the intrinsic lysosome-targeted ability that is independent of lysosomal acidity and endocytosis pathway. As a result, ASiRs show not only the stable lysosomal localization against lysosomal pH changes and negligible interference to lysosomal function, but also excellent cell-membrane-permeability due to the energy-independent passive diffusion pathway. These merits, coupled with their excellent two-photon photophysical properties, long-term retention ability in lysosomes, and negligible cytotoxicity, make ASiRs very suitable for real-time and long-term tracking of lysosomes in living cells or tissues without interference to normal cellular processes. Moreover, the easy functionalization via amino linker further allows the construction of various fluorescent probes for biological targets of interest based on ASiR skeleton, as indicated by the cancer-targeted fluorescent probe ASiR6 as well as a fluorescent peroxynitrite probe ASiR-P.

荧光技术对溶酶体的无创性和特异性可视化对于研究健康和疾病中的溶酶体运输以及评估靶向肿瘤细胞溶酶体的新型癌症治疗方法至关重要。迄今为止，存在两种基本类型的溶酶体探针，其溶酶体定位分别与溶酶体酸度和内吞途径相关。然而，前者可能遭受pH敏感的溶酶体定位和碱化诱导的溶酶体酶失活，而后者由于内吞作用过程的能量依赖性而需要较长的孵育时间才能穿透细胞膜。在这项工作中，一类新型的双光子荧光染料被称为氨基-Si-若丹明（ASiRs），具有独立的溶酶体酸性和内吞途径，具有固有的溶酶体靶向能力。结果，ASiRs不仅显示出针对溶酶体pH变化的稳定的溶酶体定位和对溶酶体功能的微不足道的干扰，而且由于与能量无关的被动扩散途径而具有优异的细胞膜渗透性。这些优点，加上其出色的双光子光物理特性，在溶酶体中的长期保留能力以及可忽略的细胞毒性，使ASiR非常适合实时和长期跟踪活细胞或组织中的溶酶体，而不会干扰正常的细胞过程。而且，经由氨基接头的容易的官能化进一步允许基于ASiR骨架构建用于感兴趣的生物靶标的各种荧光探针，如癌症靶向的荧光探针ASiR6以及荧光过氧亚硝酸盐探针ASiR-P所指示的。