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

用于 4D 核组高通量纳米显微成像的集成成像系统

• 批准号: 9003448
• 负责人: DAVID BADDELEY
• 金额: $ 33万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-30 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9003448
• 关键词: Affect; Algorithms; Architecture; Automation; Behavior; Benchmarking; Binding; Biological; Biology; Cell Nucleus; Cell physiology; Cells; Chromatin; Chromatin Fiber; Chromatin Modeling; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Color; Complex; Crowding; DNA; DNA-Binding Proteins; Data; Data Quality; Data Set; Databases; Detection; Development; Disease; Environment; Event; Feedback; Gene Expression; Generations; Genetic Transcription; Health; Histones; Hour; Image; Image Analysis; Imaging Device; Individual; Knowledge; Label; Length; Life; Malignant Neoplasms; Mammalian Cell; Maps; Measures; Mechanics; Methods; Microscope; Mission; Modeling; Molecular Conformation; Movement; Nuclear; Nucleosomes; Optics; Pathway interactions; Performance; Physics; Positioning Attribute; Procedures; Process; Proteins; Public Health; RNA; Regulation; Replication Origin; Research; Resolution; Role; Scanning; Scientist; Software Tools; Speed; Structure; Synaptonemal Complex; System; TAF3 gene; Techniques; Telomere Recombination; Testing; Thick; Time; Transcriptional Regulation; Variant; adaptive optics; base; cellular imaging; cofactor; computer infrastructure; computer science; computerized data processing; data modeling; developmental disease; embryonic stem cell; experience; flexibility; genetic information; histone modification; holistic approach; image processing; imaging system; innovation; instrument; instrumentation; interest; nanoscale; new technology; novel; particle; performance tests; piRNA; process optimization; public health relevance; repaired; single molecule; spatiotemporal; tool; tool development; transcription factor

 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.

 描述（由应用提供）：DNA，蛋白质和RNA的复合物称为染色质，是基因转录，调节，复制和修复等必需细胞过程的底物。因此，阐明其结构和动态是必不可少的，我们要了解这些过程的机制及其在发育和疾病中的影响。然而，染色质是一个难以研究的靶标：在核us内的拥挤环境中发现了染色质，在结构上是在多个长度尺度上组织的，在核内和核之间是可变的，并且具有高度动态性。捕获此信息需要仪器，该仪器可以（i）在多个长度尺度上进行测量，从整个细胞降低到数十nm，（ii）遵循活细胞中的染色质动力学，（iii）在可管理的时间范围内获取和量化数千个图像，以克服本质上的变异性并提供染色质行为的统计描述。这种仪器尚未存在，现有仪器在分辨率，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，以实现用于自动化高通量超分辨率成像和细胞核中密集的单分子跟踪的集成系统。我们的具体目的是：1）开发一种自动多色3D单分子开关（SMS）纳米镜，用于动态成像和粒子跟踪的动态成像和粒子跟踪，2）2）2）用于高通量3D- SMS纳米镜检查工具的数据处理工具，该工具的纳米纳西检查是100-1,000个细胞/h的100-1,000个细胞/h，3）均能开发出4个固定级别的固定级别的级别，这些工具是固定的，该工具是详细仪的，该工具的优势是详细范围的，这些量很详细且固定级别的级别，并获得了良好的级别。高通量粒子跟踪和SMS纳米镜检查，以及4）通过将它们应用于染色质体系结构领域的潜水员集和重要问题的潜水员集，包括转录因子和核心小体的动力学和动态，以及Synaptonemal组装和远程征膜重新组合的过程，测试并完善我们的技术发展。该提案代表了纳米镜检查图像生成程序的传统观点的基本偏差，因为动手操作过程涉及专家用户到自动化的高通量方法，重点是量化和效率。通过对成千上万个细胞可行的纳米镜检查研究，我们预计我们的仪器将首次实现核心的空间时间动力学，以定量研究到单个核病士水平。