适合蛋白质动力研究的超分辨无标记位相显微技术

In order to understand the dynamics of proteins inside and outside of cells, currently it is necessary to label both proteins and their references, such as the membrane and nuclei, for observation and analysis. Because of the large volumes and surfaces of those reference structures, photo-bleaching will cause a huge production of reactive oxide, which is toxic to cells, resulting the early deaths of cells. We suggest to use label-free methods to observe those reference structures, thus avoiding large amount of toxicity and maintaining cells’ viability. The image quality, resolution and 3D image formation of the label-free measurements should be similar to the fluorescence microscopy to guarantee high imaging speed and high quality of reference information. Current bright field microscopy and phase contrast microscopy cannot meet those requirements due to their poor resolution, contrast and artifacts. Other super-resolution phase microscopy developments either do not have 3D imaging capability, or not compatible with fluorescence microscopy. In this proposal, we will develop a super-resolution phase microscopy that has transverse resolution of ~135nm and axial resolution of ~300nm. Its 3D images are formed through z-scan and free of artifacts, thus providing high imaging speed and reliable reference information. We will use this technology to record how dendritic spines are involved in synapse plasticity； Also we will demonstrate the ability of our method to extend imaging time of mitochondria in living cells by ~10x. .The proposed instrument will have significant impacts in basic biology, and in related fields such as medicine and pharmacology. In particular, it will help to (i) extend the time points of live cell for study (ii) provide more reliable information when exogenous agents are suspected of affecting cell functions (iii) provide dynamic and detailed structural information besides pathways of protein and vesicles (iv) observe fast dynamics of fragile cellular structures such as dendritic spines.

为了理解细胞内外蛋白质的运动，不仅需要对蛋白质本身进行荧光标记，对其运动的参照物如细胞膜细胞核等也要标记。对这些参照物的大面积标记将导致细胞由于光漂白和光毒而提前死亡。常规无标记技术如明光和相衬，由于分辨率、对比度和伪影，不能提供高质量的参照物信息。 目前发展的一些超分辨率位相技术也由于成像方式和速度等问题不足以协助荧光模式 。 针对这一现状，我们将利用非相干结构光照明发展超分辨率位相显微成像理论和技术，实现横向分辨率~135纳米，纵向分辨率~300纳米。且三维成像的方式跟荧光模式一样，不受激光散射斑的影响，保证成像速度和参照物信息的准确性。我们将应用该技术（1）观察树突棘参与突触可塑性的过程；（2）将线粒体的动力学观察时间延长~10倍。该工作将为蛋白质等微纳细胞结构的动力学提供方法和技术支撑。

针对活细胞长时间观察的要求，本项目计划开展以下两个方面的研究：在系统搭建方面，搭建一个利用非相干光照明的无标记显微系统，旨在提高当前位相技术的分辨率；在应用方面，利用此技术观察一些细胞的动态。课题资助金额为25万，起止时间为2017.1.1-2018.12.31，执行时间共两年。目前项目进展顺利。已发表文章1篇，2篇在投。.目前第一代多模态显微系统已完成搭建，包括一个无标记位相通道和一个荧光通道。位相通道的分辨率，对比度和成像速度都优于国际同行，利用此系统，我们不仅能观察到线粒体的整体形状，也首次在无标记的情况下能看到线粒体的内部结构以及其融合和分裂的过程，内质网网络的清晰结构和振动。同时我们也看到了从未报道过的线粒体自旋现象。在突触应用上，由于神经细胞本身非常脆弱， 对环境要求比较苛刻，不适合搬运。因此目前还没有开展这方面的应用。