Enabling Nanoscale Dynamic Imaging of Vesicles and Organelles

 该项目的长期目标是使活细胞内的囊泡和细胞器动态成像具有前所未有的空间和时间分辨率。新的超分辨率技术，如单分子切换（SMS）纳米显微镜的最引人注目的优势是潜在的图像动态过程中的活细胞与10-20 nm的分辨率，从而解决了许多悬而未决的问题，在细胞生物学需要高的结构和时间分辨率。其中一个问题是高尔基体是如何动态地组织起来的，这是一个主要的和高度争论的谜。虽然固定细胞中的SMS成像已经产生了令人印象深刻的新生物学发现，但解决动力学的潜力还很远。 从完全实现。主要限制因素包括：（i）缺乏既能达到最高分辨率又能在与活细胞中的扩展成像相容的环境和速度下达到最高分辨率的仪器，（ii）缺乏能以高特异性、密度和亮度在活细胞内无毒标记和转换的良好探针，以及（iii）关于如何处理高速超分辨率显微镜提供的潜在不完整数据的不确定性。出于一个长期存在的生物学问题，即蛋白质通过高尔基复合体被贩运的机制，我们建议解决这些主要的当前限制，并开发显微镜硬件，探针和算法，使动态纳米显微镜成为现实。我们的具体目标是：1）实现4Pi-SMS仪器，其将以最小的光损伤在活细胞中提供最佳的3D分辨率，2）开发一种新类型的可闪烁的高密度活细胞SMS探针以成像高尔基体，以及3）开发新的图像处理工具，其利用先前的生物物理学知识来改善高尔基体形态的重建和量化。这三种方法的发展将被应用到一个新的合成生物学系统，“landlocks”高尔基池线粒体，并将促进有利的几何形状monito高尔基功能。虽然针对高尔基体，我们的方法的发展将广泛适用于活细胞超分辨率动态成像的细胞内几乎每一个细胞器。

 DESCRIPTION (provided by applicant): The long-term aim of this project is to enable the imaging of vesicle and organelle dynamics inside living cells with unprecedented spatial and temporal resolution. The most compelling advantage of new super-resolution techniques such as single molecule switching (SMS) nanoscopy is the potential to image dynamic processes in living cells with 10-20 nm resolution and hence solve the many open questions in cell biology which need both high structural and temporal resolution. One such problem is how the Golgi is dynamically organized, a major and highly debated enigma. Although SMS imaging in fixed cells is already yielding impressive new biological discoveries, the potential to resolve dynamics is far from fully realized. Key limiting factors include: (i) lack of instrumentation capable of both attaining the highest resolutions and doing so in an environment and at a speed which are compatible with extended imaging in living cells, (ii) lack of good probes which can non-toxically label and switch inside a live cell with high specificity, density and brightness, and (iii) uncertainty about how to deal with the potentially incomplete data that high-speed super-resolution microscopy delivers. Motivated by a long-standing biological problem, namely the mechanism by which proteins are trafficked through the Golgi complex, we propose to address these major current limitations and develop the microscope hardware, probes and algorithms to make dynamic nanoscopy a reality. Our specific aims are: 1) To implement a 4Pi-SMS instrument which will deliver the best possible 3D resolution in living cells with minimal photodamage, 2) To develop a new genre of blinkable high-density live-cell SMS probes to image the Golgi, and 3) To develop new image processing tools which leverage prior biophysical knowledge to improve the reconstruction and quantification of Golgi morphology. These three methodological developments will be applied to a novel synthetic biology system that 'landlocks' Golgi cisternae to mitochondria and will facilitate favorable geometries to monito Golgi function. Although targeted at the Golgi, our methodological developments will be broadly applicable to live-cell super-resolution dynamic imaging of nearly every organelle within the cell.