NO是生物体内起重要生理作用的活性氮，在心血管系统、免疫系统、人体神经系统中起着十分重要的作用。然而，异常水平的NO与多种疾病密切相关。因此，近年来，生物体内NO的检测越来越受到关注。其中，有机荧光探针具有灵敏度高、操作简便、膜通透性好和原位检测等众多优点而得以广泛应用。但报道的大多是单光子荧光探针，此类探针在生物成像时具有容易产生光漂白、光致毒及组织穿透深度有限等缺点。而双单光子荧光探针作为分子工具在生物学和疾病诊断等方面使用能克服以上缺点。到目前为止，线粒体靶向NO双光子比率小分子荧光探针还未见文献报道。本项目设计了一系列适合发展靶向比率荧光探针的双光子荧光共振能量传递染料；再在其上修饰特异性的NO反应位点和线粒体靶向季鏻盐，合成线粒体靶向NO双光子比率荧光探针；并应用于活细胞和组织中进行双光子激发荧光成像。这种设计有望提出一种线粒体靶向比率荧光探针运用于生物中NO检测的新方法。

Nitric oxide (NO), a biologically significant reactive nitrogen species (RNS), play a critical role in cardiovascular system, immune system, the central and peripheral nervous system. However, when the cells cannot maintain the level within the physiological range, NO is engaged in some diseases, such as stroke, heart disease, hypertension, neurodegeneration, erectile dysfuction, and gastrointestinal diseases. Therefore, it is of high importance to detect NO in living systems in recent years. Because the fluorescent probe has many advantages including high sensitivity, simple operation, good reproducibility, good membrane permeability and detection in situ, fluorescent probes play an irreplaceable role in the detection of NO in vivo. However the single photon fluorescence probe reported generates light toxicity, bleaching and limited tissue penetration depth,two-photon fluorescence probe is more suitable to be used as molecular tools in biology and disease diagnosis。So far, mitochondria-targetable NO two-photon ratiometric fluorescent probes have not been reported. In this project we have designed a series of novel dyes based on fluorescence resonance energy transfer, which are suitable for development the targetable two-photon ratiometric fluorescence NO probe, and take it as the platform, modified specificial reaction site of NO and the mitochondrial targeting orgnic small molecules to develop the mitochodria-targetable two-photon ratiometric fluorescence probe for NO. This work should open a new avenue to develop organell-targetable two-photon ratiometric fluorescence probes.