利用STORM和FLIM方法对基因组DNA压缩进行多模态光学表征

It is well known that genomic DNA compaction is involved in a series of essential cellular physiological processes, including gene activation as well as DNA replication and repair. However, there is not any appropriate real-time dynamic optical method of monitoring the DNA compaction process in a high-resolution and quantitative way. Here, we provide a multimodal super-resolution stochastic optical reconstruction microscopy (STORM)/fluorescence lifetime imaging microscopy (FLIM) optical technique. STORM technique can provide a resolution of ~50 nm of fluorescent dye labeled chromatin fibers. In parallel, FLIM modality will be used to analyze proximity between labelled DNA strands within 1-10 nm, through the Förster Resonance Energy Transfer (FRET) mechanism. In the outcome, STORM and FLIM-FRET data sets will be mutually combined to enhance the resolution capabilities of nanoscopic imaging. With the development of the combined multimodal system, we anticipate that this STORM-FLIM super-resolution imaging will enable dynamic analysis and characterization of compacted and open chromatin structures within different stages of cell cycle. Also, we will specifically address differences in compaction between chromatin containing active genes and chromatin containing non-coding DNA sequences. Furthermore, we will apply these novel optical techniques in probing the single strand DNA in cell nucleus. In conclusion，this project will establish a new optical multimodal platform for monitoring genomic DNA compaction in a quantitative way, providing novel optical microscopy tools for quantitative studies of chromatin compaction and gene activation. These developments will be highly demanded in a broad area of quantitative cell science and applied biomedicine.

基因组DNA压缩参与基因激活、基因复制和修复等多个重要的细胞生理学过程，但目前缺乏可对该过程进行高空间分辨、实时动态以及定量表征的光学方法。本项目提出并发展一种基于超分辨随机光学重构显微（STORM）和荧光寿命显微成像（FLIM）联用的基因组DNA压缩多模态光学表征方法，利用STORM方法获得荧光标记的染色质丝超分辨图像，利用基于FLIM的荧光共振能量转移（FRET）方法分析荧光标记DNA双链的相互作用，两种成像方法的结合联用将提高对DNA压缩的光学表征能力。在此基础上，利用该光学方法和平台监测和表征细胞周期不同时间染色质压缩和打开的动态变化过程，分辨染色质富含活性基因和非编码序列区域DNA压缩的不同之处，建立在细胞核中检测单链DNA位点的新型光学方法。本项目研究将建立对染色质压缩进行定量分析的STORM/FLIM多模态光学方法，对活细胞中DNA定量研究及基因活性监测具有重要科学意义。

细胞是生命活动的基本单位，目前细胞生物学中一个研究热点是实时监测基因组DNA压缩如何参与基因激活、基因复制和修复等多个重要的细胞生理学过程，但目前缺乏可对该过程进行高空间分辨、实时动态以及定量表征的光学方法。针对该问题，本项目提出了一种基于超分辨随机光学重构显微（STORM）和荧光寿命显微成像（FLIM）联用的基因组DNA压缩多模态光学表征方法；利用STORM方法获得荧光标记的染色质丝超分辨图像，理解细胞周期中染色质丝的变化情况；提出了利用基于FLIM的FRET方法分析荧光标记DNA双链的相互作用，为在单细胞水平研究DNA双链提供了一种基于荧光显微成像方法的单细胞研究方法；两种成像方法的结合联用可在活细胞内实现对DNA压缩的超分辨光学表征。.与传统的生化检测及细胞生物学研究方法相比，本方法具有实时动态、无损、灵敏度高、分辨率好等优点，达到了本项目预期的单细胞的染色质定量检测等应用目标，并为研究活细胞的细胞核内染色质压缩提供了一种通用的定量分析的STORM/FLIM多模态光学方法，有望在细胞生物学机制研究特别是活细胞中DNA定量研究及基因活性监测得到广泛应用。.综上所述，本项目具有较好的研究意义和应用价值。本项目研究成果在国内外优秀专业杂志上发表研究论文5篇，其中4篇为SCI收录；申请并获授权实用新型专利 1 项；参加国际学术会议3次；培养博士后2名、硕士研究生 2名。