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

Overview: This is a TYPE A proposal to develop a high temporal and spatial imaging instrument whose purpose is to combine high throughput single-molecule spectroscopy (TIRF/FCS) into a single cost-effective instrument controlled by a single software platform. The proposed research will benefit the following biological research community: molecular, physical and cellular biosciences. The use of single-molecule fluorescence spectroscopy and microscopy has greatly expanded our capacity to measure biochemical and cellular processes in vivo at the molecular level. However, they are currently available as separate instruments that are not only costly, but also cumbersome to operate. In this proposal we aim to develop a unique instrument that can be reconfigured on the fly for multi-modality imaging in a single device with high throughput capabilities. Our new instrument, together with newly developed data collection and analysis software, will provide the ability to correlate data on single samples between imaging modes, therefore enabling new research capabilities to extract dynamics from single biological molecules in vitro and in cells. The ability to resolve structural rearrangements, biomolecular interactions, and binding kinetics of complex systems is essential to gaining crucial insights into the inherently heterogeneous and complex nature of biology. Intellectual Merit: The outcomes of this proposal will include the development of an instrument capable of rapidly transitioning between TIRF and FCS/FCCS with high throughput capabilities. The hardware and software platforms will allow multi-modal detection of samples. Using designed hardware and customized control software, the proposed instrument will have the unique feature to switch rapidly between multiple ensemble and single molecule imaging modalities on the fly. This can be applied to isolated biomolecules, single cells, or tissue samples. Our design will be able to function in multiple modes including those with parallel detection of multiple molecules and then those that require high temporal resolution of a single molecule. This versatility is necessary to account for the diversity of experimental parameters required for biological imaging. Specific imaging modes include fluorescent correlation and cross-correlation spectroscopy (HT-FCS/FCCS), HT-TIRF-FCS, and FRET. Due to the innovative, multi-modal capabilities of the instrument, we will also develop a new open-source fluorescence spectroscopy analysis package, Open Fluorescence Spectroscopy (OpenFS). In addition to providing the analysis capability for our new multi-modal data, we envision that such an open-source package will eventually lead to reusable and standard software modules, which could compete or replace the currently fragmented and often proprietary analysis software. Broader Impacts: The proposed research enabled by our instrument spans the disciplines of chemistry, physics, molecular biology, and engineering to answer fundamental questions in biology. Its multidisciplinary nature will create unique training opportunities for undergraduate and graduate researchers. The presence of state-of-the-art imaging capabilities at Kentucky will greatly enhance the capability of performing cutting-edge research by researchers and students in this region. To the best of our knowledge most of the individual methods are unavailable within the region let alone the novel capability to switch between single molecule, ensemble imaging, and super resolution technologies within a single instrument. The substantial involvement of the PIs and senior personnel in a broad range of outreach programs, including NSF-funded REUs and the Cancer Nanotechnology Training Center (CNTC) programs, will ensure involvement of undergraduate students. The developed analysis package OpenFS 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. In addition, we will utilize the Research Media Internship program at the VisCenter to produce media documenting the progress of this project. Through these broad distribution channels, the visual media can be a driving force to attract young students for STEM subjects early in their studies.