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

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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.