基于随机光学涨落技术的活细胞内线粒体动态与功能成像研究

Real-time monitoring the dynamic changes of fine structure in living cells by super-resolution fluorescence microscopy has become an increasingly imminent problem to be solved in the study of dynamic and functional of subcellular structures, protein dynamics and biomolecular imaging. However, due to the intrinsic trade-off between the spatial and temporal resolutions, most existing super-resolution microscopy methodologies have problems of poor time resolution, high excitation light intensity, and complex system structure, which make it difficult for imaging of live cells. In this project, with development of optimization algorithm and blinking fluorescent probes which are suitable for fast living cell microscopy, the application of super-resolution optical fluctuation imaging (SOFI) technique in the dynamic and functional imaging of mitochondria in living cells will be implemented. The main contents are as follows: First, A deconvolution optimization in covariance imaging for super-resolution based on standard deviation will be implemented to enhance the quality and imaging-speed of reconstructed image; Then, A Cy series fluorescent probes with blinking enhanced will be developed to obtain dynamic SOFI results in living cells with ultra-high lateral resolution of <60 nm at temporal resolutions of 0.1 ms; At last, the specific applications of this system to image the fusion and division dynamic of mitochondria in real-time will be implemented to explore the physiological function of mitochondria’s fusion and division activity in maintaining cell’s activity. In short, this project is expected to provide an important measure for the analysis of the working mechanism within the subcellular apparatus.

应用超分辨荧光显微术实时监测活细胞内精细结构的动态变化是当前研究亚细胞器动态与功能、蛋白质动力学等成像领域迫切需要解决的科学问题。然而由于时空分辨之间相互制约，现有的超分辨显微术存在成像速率慢、激发光强度大和系统结构复杂等问题，难以对活细胞进行动态检测。本项目拟基于超分辨光学涨落成像(SOFI)方法，通过优化算法和开发适用于活细胞内动态显微的闪烁荧光探针，实现利用SOFI技术实时监测线粒体的动态与功能成像。主要研究内容有：研发一种基于标准差的解卷积优化超分辨协方差成像算法，提高重构图像的质量和成像速率；开发一种以Cy系列基团为母体的闪烁加强荧光探针，实现活细胞内横向<60nm和0.1s的超高时空分辨动态SOFI成像；将SOFI技术应用于实时监测线粒体融合分裂进程，并探索线粒体融合分裂活性在保持细胞活跃方面的生理意义。本项目的研究旨在为解析亚细胞器内部工作机理提供重要的检测手段。

应用超分辨荧光显微术实时监测活细胞内线粒体的动态变化对于研究其生理机制和功能具有重要意义。本项目基于随机光学涨落成像技术（SOFI）的新方法实现快速的活细胞内线粒体动态纳米分辨成像。研究内容包括：研发一种解卷积优化超分辨协方差成像算法，提高重构图像中样品结构的完整性及成像速率，实现了重构20帧原图，获得50nm的横向分辨率和0.4s的时间速率；与深圳大学合作开发一种新型的Cy系列荧光探针，不需要使用缓冲液体系及对细胞有害的含巯基试剂，在低功率单束激光照射下产生光致闪烁，适用于活细胞的超分辨成像；应用新的SOFI技术对活细胞内线粒体的动态过程成像，实现了重构25帧原图可获得横向<100nm的分辨率，时间速率为0.1s，可观察到线粒体融合分裂的动态过程。本项目的研究可为解析线粒体的工作机理提供重要的检测手段。