An Integrated Imaging System for High-throughput Nanoscopy of the 4D Nucleome

 描述（由申请人提供）：称为染色质的DNA、蛋白质和RNA复合物是基因转录、调节、复制和修复等基本细胞过程的底物。因此，解开它的结构和动力学是必不可少的，如果我们要了解这些过程的机制及其在发展和疾病的影响。然而，染色质是一个难以研究的目标：它在细胞核内的拥挤环境中被发现，在结构上以多个长度尺度组织，在细胞核内和细胞核之间是可变的，并且是高度动态的。捕获这些信息需要仪器，其可以（i）在多个长度尺度上测量，从整个细胞到几十nm，（ii）跟踪活细胞中的染色质动力学，以及（iii）在可管理的时间范围内获取和量化数千个图像，以克服固有的可变性并提供染色质行为的统计描述。这样的仪器还不存在，现有的仪器在分辨率、动态速度和吞吐量方面受到限制。我们提出了一种创新的多学科方法，该方法结合了光学，数据处理和建模的发展，以实现自动化高通量超分辨率成像和细胞核中密集单分子跟踪的集成系统。我们的具体目标是：1）开发用于核中的动态成像和颗粒跟踪的自动化3D单分子切换（SMS）纳米镜，2）开发用于100- 1，000个细胞/小时的高通量3D-SMS纳米镜的数据处理工具，3）开发染色质建模工具，利用高通量粒子-图像分析提供的4D数据的前所未有的细节水平和统计深度。跟踪和SMS纳米显微镜，和4）测试的性能和完善我们的技术发展，通过将它们应用于染色质结构领域的一系列代表性和重要的问题，包括转录因子和核小体的流动性和动力学，以及联会装配和端粒重组的过程。该提议代表了从纳米显微镜图像生成过程的传统观点的根本偏离，该传统观点是将大量涉及专家用户的动手过程视为专注于定量和效率的自动化高通量方法。通过对数万个细胞进行纳米显微镜研究，我们预计我们的仪器将首次使核组的时空动力学能够定量研究到单个核小体水平。

 DESCRIPTION (provided by applicant): The complex of DNA, protein, and RNA known as chromatin is the substrate for essential cellular processes such as gene transcription, regulation, replication, and repair. Unravelling its structure and dynamics is therefore essential f we are to understand the mechanics of these processes and their effects in development and disease. Chromatin is, however, a difficult target to study: it is found in a crowded environment within the nucleus, is structurally organized on multiple length scales, is variable within and between nuclei, and is highly dynamic. Capturing this information requires instrumentation which can (i) measure on multiple length scales, from the whole cell down to tens of nm, (ii) follow chromatin dynamics in living cells, and (iii) acquire and quantify thousands of images in a manageable time frame to overcome the intrinsic variability and provide a statistical description of chromatin behavior. Such an instrument does not yet exist, with existing instrumentation being limited in resolution, dynamic speed, and throughput. We propose an innovative multi-disciplinary approach that combines developments in optics, data processing and modeling to realize an integrated system for automated high-throughput super- resolution imaging and dense single-molecule tracking in the cell nucleus. Our Specific Aims are: 1) Develop an automated multicolor 3D single-molecule switching (SMS) nanoscope for dynamic imaging and particle-tracking in the nucleus, 2) Develop data processing tools for high-throughput 3D- SMS nanoscopy of 100-1,000 cells/h, 3) Develop chromatin modeling tools that take advantage of the unprecedented level of detail and statistical depth of the 4D data provided by high-throughput particle- tracking and SMS nanoscopy, and 4) test the performance and refine our technical developments by applying them to a diverse set of representative and important questions in the field of chromatin architecture, including the mobility and dynamics of transcription factors and nucleosomes, and the processes of synaptonemal assembly and telomere recombination. The proposal represents a fundamental departure from the traditional view of the nanoscopy image generation procedure as a hands-on process heavily involving an expert user to an automated, high- throughput method with focus on quantification and efficiency. By making nanoscopy studies of tens of thousands of cells feasible, we anticipate that our instrument will enable, for the first time, the spatiotemporal dynamics of the nucleome to be quantitatively investigated down to the single nucleosome level.