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

肯塔基大学被授予开发一种用于分析生物分子相互作用的新成像仪器。高分辨率荧光技术的发展对生物化学和细胞过程的理解大有裨益。全内反射显微镜(TIRF)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，已成为研究膜蛋白的标准技术。荧光相关光谱(FCS)提供了在体外和细胞内从单个生物分子中提取动力学的能力，从而对它们内在的异质性和复杂性产生了至关重要的见解。这些技术的主要局限性之一是，它们是高度劳动密集型的，因此主要局限于单样本分析，其次是昂贵的人工数据处理。拟议的仪器将通过在单一、成本效益高的成像和数据收集平台下开发这两种技术的高吞吐量版本来克服这些挑战。此外，将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)将是开源的，并可在网上免费获得，为通常与特定设备捆绑在一起的昂贵且专有的成像软件提供了替代方案。这台仪器的研究跨越了化学、物理、分子生物学和工程学等学科，以回答生物学中的基本问题。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本科学发现。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。解决复杂体系的结构重排、生物分子相互作用和结合动力学的能力对于跨越生物、化学、工程和物理等多个学科的研究目标至关重要。对其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是必不可少的。该项目的目标是开发一个独特的、多功能的、多方面的平台，以扩展高通量TIRF和FCS的高时间和空间成像。该仪器将能够在多种高通量模式下工作，并提供在聚合物、重组或细胞系统中使用系综和单分子荧光测量来提取广泛生物分子动力学的工具。由于该仪器的多模式能力，将开发一个新的开放源码荧光光谱分析包，开放荧光光谱(OpenFS)，提供可重复使用的软件模块，用于处理荧光光谱数据。该仪器将以经济高效和用户友好的形式进行高通量、高灵敏度的研究，有可能改变生物分子动力学的研究，并允许将TIRFM和FCS扩展到以前无法访问的应用。

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.