超分辨成像荧光染料分子的高通量筛选及发光机制定量研究

Super-resolution fluorescence imaging has become one of the most powerful techniques in the cutting-edge studies of biological science. Fluorescent molecules such as organic dyes play a key role in this field, as their performance determines the quality and fineness of super-resolution images. One of the difficulties is the limited number of fluorescent dyes with good photo-physical properties available for the super-resolution imaging. On the current stage, there is no cost-effective and efficient way of screening and evaluating the performance of different fluorescent dyes in parallel and in details. In addition, there is lack of fine control over the micro-environment where the dye molecules are located, which significantly influences dyes’ performance in the imaging process. In order to tackle these problems, the applicant proposes to develop a high-throughput microfluid platform for the super-resolution imaging, which can be used to precisely control the micro-environment of dye molecules, to screen and evaluate their fluorescence properties effectively and in parallel. In the meanwhile, with the help of such high-throughput microfluid platform, the applicant hopes to clarify the influence of redox micro-environment on the fluorescence of dyes on the single molecule level, and points out the key factors which help improve the quality of live cell imaging. The final goal is to move forward the applications of fluorescent dyes in the super-resolution imaging field.

超分辨荧光成像是当前生命科学研究中不可或缺的技术手段，荧光分子在该技术应用中起着重要作用，决定了成像的质量与精细度。原则上具备亮暗态光转换的荧光染料分子都有超分辨应用潜力，但实际上可用的染料分子种类非常有限。究其原因，一是缺乏大规模系统筛选和评估染料分子光学性质的技术方法，二是缺乏对影响分子发光微环境的精确调控。为解决以上超分辨领域的重要问题，申请人拟构建一个具有微环境精细控制功能的高通量筛选微流控平台，精确控制荧光染料分子在超分辨成像过程中的氧化还原微环境，系统、高效地对染料分子超分辨能力进行筛选与评估，拓展应用于超分辨成像的荧光染料分子库。同时，在单分子水平上，定量研究氧化还原微环境影响荧光染料分子发光的机理，阐明提高活细胞超分辨成像质量的关键要素，推进超分辨荧光成像在生物医学领域的广泛应用。

近年来，超分辨荧光成像已经成为高水平生命科学研究中不可或缺的工具，而荧光分子的光物理化学性质决定了成像的质量。通过项目的资助，我们构建了一个具有微环境精细控制功能的高通量微流控筛选平台，其核心功能包括：对化学微环境的精细控制部分以及对气体微环境的控制部分，利用该平台，我们实现了对具有潜力应用于超分辨成像的荧光分子进行有效地筛选，研究成果已经申请了5项专利，其中1项已经授权。另外，在单分子水平上，我们系统的探索了三类荧光染料的发光机制及超分辨成像能力，通过将染料进行探针标记，实现了对多种活细胞亚结构（线粒体、溶酶体、细胞核组蛋白等）的超高分辨率成像，研究成果形成4篇科研论文，其中2篇已经发表在化学类顶级期刊Journal of the American Chemical Society杂志上，1篇发表篇在Analytical Chemistry。以上工作，指明了提高活细胞超分辨成像质量的关键要素，实现了具有良好时间/空间分辨率的高质量活细胞超分辨成像。本项目不仅进一步扩展了超分辨荧光染料库，而且高质量的推进了超分辨荧光成像在生物医学领域的广泛应用。