IDBR: Type A: Development of a high throughput total internal reflection and fluorescence correlation platform for analysis of biomolecular interactions

IDBR：A 型：开发用于分析生物分子相互作用的高通量全内反射和荧光相关平台

• 批准号: 1556281
• 负责人: Jason DeRouchey
• 金额: $ 58.93万
• 依托单位: University of Kentucky Research Foundation
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1556281&HistoricalAwards=false
• 关键词: IDBR; Type; Development; throughput; total

An award is made to the University of Kentucky to develop a new imaging instrument for analysis of biomolecular interactions. Understanding of biochemical and cellular processes has profited enormously from the development of high resolution fluorescence techniques. Total internal reflection (TIRF) microscopy is a technique that allows high resolution imaging of a thin layer of the cell close to the plasma membrane and has become a standard technique to study membrane proteins. Fluorescence correlation spectroscopy (FCS) provides the capability to extract dynamics from single biological molecules in vitro and in cells yielding crucial insights into their inherently heterogeneous and complex nature. One of the primary limitations of these techniques is that they are highly labor intensive and thus have been primarily restricted for single sample analysis followed by costly manual data processing. The proposed instrument will overcome these challenges by developing high throughput versions of both techniques under a single, cost-effective imaging and data collection platform. In addition, a new analysis package Open Fluorescence Spectroscopy (OpenFS), to be developed by researchers and computer scientists, will be open-source and made freely available online, providing an alternative to costly and proprietary imaging software that is often tied to a particular device. Research enabled by this instrument spans the disciplines of chemistry, physics, molecular biology, and engineering to answer fundamental questions in biology. The ability to perform multiple and single molecule studies at high throughput has the capacity to accelerate basic scientific discoveries essential to the biosciences. The multidisciplinary nature of this project will also create unique training and educational opportunities for undergraduate, graduate and post-doctoral researchers. The ability to resolve structural rearrangements, biomolecular interactions, and binding kinetics of complex systems is essential to the research aims across multiple disciplines of study including biology, chemistry, engineering, and physics. Imaging of fluorescent biomolecules in their natural cellular environment to obtain quantitative information about their dynamic behavior is essential for further understanding their roles in cellular processes. The goal of this project is to develop a unique, versatile, and multi-faceted platform that will extend high temporal and spatial imaging for high throughput TIRF and FCS. The instrument will be able to function in multiple high throughput modes and provide the tools to extract a broad range of biomolecular dynamics using ensemble and single-molecule fluorescence measurements in polymeric, reconstituted, or cellular systems. Due to the multi-modal capabilities of the instrument, a new open-source fluorescence spectroscopy analysis package, Open Fluorescence Spectroscopy (OpenFS), will be developed offering offers reusable software modules for processing of fluorescence spectroscopy data. The instrument will conduct high-throughput, high sensitivity studies in a cost-effective and user friendly format that has the potential to transform studies of biomolecular dynamics and allow the extension of TIRFM and FCS to applications previously inaccessible.

肯塔基大学获得了开发一种用于分析生物分子相互作用的新型成像仪器的奖项。 高分辨率荧光技术的发展使人们对生化和细胞过程的理解受益匪浅。全内反射（TIRF）显微镜是一种可以对靠近质膜的细胞薄层进行高分辨率成像的技术，并且已成为研究膜蛋白的标准技术。荧光相关光谱 (FCS) 能够从体外和细胞内的单个生物分子中提取动力学，从而对其固有的异质性和复杂性产生重要的见解。这些技术的主要局限性之一是它们是高度劳动密集型的，因此主要局限于单个样本分析，然后是昂贵的手动数据处理。 所提出的仪器将通过在单一、经济高效的成像和数据收集平台下开发这两种技术的高通量版本来克服这些挑战。此外，由研究人员和计算机科学家开发的新分析包开放荧光光谱（OpenFS）将开源并免费在线提供，为通常与特定应用程序绑定的昂贵且专有的成像软件提供了替代方案。设备。 该仪器支持的研究跨越化学、物理、分子生物学和工程学学科，以回答生物学的基本问题。 以高通量进行多分子和单分子研究的能力能够加速对生物科学至关重要的基础科学发现。 该项目的多学科性质也将为本科生、研究生和博士后研究人员创造独特的培训和教育机会。 解决复杂系统的结构重排、生物分子相互作用和结合动力学的能力对于生物学、化学、工程学和物理学等多个学科的研究目标至关重要。对自然细胞环境中的荧光生物分子进行成像以获得有关其动态行为的定量信息对于进一步了解它们在细胞过程中的作用至关重要。 该项目的目标是开发一个独特、多功能和多方面的平台，为高通量 TIRF 和 FCS 扩展高时间和空间成像。 该仪器将能够在多种高通量模式下运行，并提供使用聚合物、重构或细胞系统中的整体和单分子荧光测量来提取广泛的生物分子动力学的工具。 由于该仪器的多模式功能，将开发一种新的开源荧光光谱分析软件包，即开放荧光光谱（OpenFS），提供用于处理荧光光谱数据的可重复使用的软件模块。该仪器将以经济高效且用户友好的方式进行高通量、高灵敏度研究，有可能改变生物分子动力学研究，并将 TIRFM 和 FCS 扩展到以前无法访问的应用。