以CRISPR-Cas9为代表的新型基因编辑技术可在活细胞中特异性识别及编辑基因，在基因治疗、物种改良等方面具有重大意义和经济价值，但目前缺乏对活细胞中基因编辑过程进行纳米分辨和定量表征的合适的实时动态光学方法。本项目提出并发展一种受激发射损耗（STED）显微成像和双光子激发荧光寿命显微成像（FLIM）相结合的基因编辑多模态光学表征方法，利用STED方法获得荧光标记靶DNA超分辨结构信息，利用FLIM-FRET（荧光共振能量转移）方法获得Cas9等内切酶活性信息。两种成像方法联用，可实现活细胞基因编辑过程中核酸内切酶作用区域的生物分子和微环境变化的动态监测，从而为基因编辑效率的优化提供一种快速、高效的检测方法。本项目将建立一种对基因编辑进行活细胞定量分析的STED/FLIM多模态光学方法，在活细胞基因编辑研究领域具有重要科学意义和应用价值，并有望在生命科学定量分析领域获得广泛应用。

New gene editing techniques represented by CRISPR-Cas9 can efficiently recognize and edit the target genes in living cells, is of great scientific significance and economic value in the fields of gene therapy, genetic improvements of species and so on. However, there is a lack of established optical techniques for real-time dynamic analysis of gene editing process in living cells in a high-resolution (nano-scale) and quantitative way. Here, this project provides a multimodal super-resolution stimulated emission depletion (STED) microscopy combined with a two-photon excited fluorescence lifetime imaging microscopy (FLIM) optical technique for monitoring gene editing. STED technique can provide a resolution of ~50-100 nm of fluorescent dye labeled DNA. Meanwhile, FLIM modality will be used to analyze the activities of Cas9 enzymes as well as to monitor the interaction between enzymes and labeled DNA strands within 1-10 nm, through the fluorescence resonance energy transfer (FRET) method. As a result, STED and FLIM-FRET data sets will be mutually integrated to improve the resolution capabilities of nanoscopic imaging of bio-molecules and to monitor micro-environmental changes in the area of gene editing. Moreover, the development of this STED/FLIM super-resolution imaging method can provide a fast and efficient detection method for further optimization of gene editing efficiency. In conclusion, this project will establish a new optical STED/FLIM multimodal optical platform for quantitative analysis of gene editing process in live cells. The establishment of this method has important significance and application value in the field of gene editing inside living cells, which is also highly demanded in a broad area of quantitative analysis of life sciences.