Two-photon (TP)-based ratiometric fluorescence probe has become an efficient and attractive technique for fluorescence imaging studies of biological events. Therefore, construction of some new type functional TP ratiometric fluorescent dyes has attracted great attention. In this work, we adopted an optically superior TP fluorophore to develop a TP ratiometric semi-rhodamine "switch" fluorescent dye platform (KzRh), and a peroxynitrite anion (ONOO⁻) fluorescent probe (KzRhONOO) with TP excitation were constructed based-on this platform for detecting ONOO⁻ in living cells and zebrafish, which exhibits a target-modulated ratiometric TP fluorescence response through the semi-rhodamine's close/opene form of the spirocycle. Such a scaffold affords KzRhONOO possesses dual-emission peak separated by 50 nm, rapid response (<15.0 s), high selectivity and sensitivity with a detection limit is as low as 10.9 nM for ONOO⁻, and as well as low cytotoxicity. Moreover, experiments showed that the KzRhONOO is a reliable and specific probe for detecting ONOO⁻ in living cells and zebrafish with dual-channel (green and red channel, respectively). Based on the above results, we hope the new platform could construct many different types of TP ratiometric fluorescent probes for bioimaging research.

基于双光子（TP）的比例荧光探针已成为一种用于生物学事件荧光成像研究的有效且有吸引力的技术。因此，一些新型功能性TP比例荧光染料的构建引起了极大的关注。在这项工作中，我们采用的光学优于TP荧光团开发TP比例半罗丹明“开关”荧光染料平台（KzRh）和过氧亚硝酸盐阴离子（ONOO ^- ）荧光探针（KzRhONOO）与TP激发构建基于上这个用于检测ONOO的平台^-在活细胞和斑马鱼中，它通过螺环的半若丹明的闭合/开放形式表现出目标调节的比例TP荧光响应。这样的支架，得到KzRhONOO具有双发射峰分离由50nm时，快速反应（<15.0 S），高的选择性和灵敏度与检测限低至10.9纳米的ONOO ^- ，和以及低的细胞毒性。此外，实验表明，KzRhONOO是用于检测ONOO可靠且特异性探针^-在活细胞和斑马鱼与双通道（绿色和红色信道，分别地）。基于以上结果，我们希望新平台能够构建用于生物成像研究的许多不同类型的TP比例荧光探针。