SBIR Phase II: Time-Resolved Fluorescence (TRF) Microscopy of Live Cells with Cell-Penetrating Peptides and Other Targeting Agents

This Small Business Innovation Research (SBIR) Phase II project addresses challenges found with the current methods of microscopic imaging of protein-protein interactions in living cells. These methods rely extensively on Förster Resonance Energy Transfer (FRET) between cyan (CFP) and yellow fluorescent proteins (YFP). These methods are problematic, due to the interference from background noise and the intrinsic photophysical properties of these fluorophores. We have developed a system that uses a lanthanide complexdonor in combination with time resolved fluorescent microscopy, which overcomes these limitations. The research objectives of this project are to expand the capability of these luminescent probes, as well as scale-up in order to provide enough material to meet manufacturing needs for initial productsales. By the end of the project, we will have reagents for performing site directed time-resolved measurements in live cells and an operational prototype time-resolved imaging module. The broader impact/commercial potential of this project, if successful, is the potential to develop a new class ofcell imaging reagents and techniques. This innovation will improve the ability of researchers to follow protein-protein interaction pathways with quantitative accuracy that has not been available before. This will impact not only fundamental and applied research but also primary healthcare throughthe discovery of novel pharmaceutical targets and mechanisms to diagnose and treat disease. The design and use of novel probes to study structure and function at the molecular and subcellular level in living cells is a topic of great importance, with a growing need for new approaches and tools to visualize not only the distribution of molecular species in cells, but the manner in which they interact. Protein-protein interactions and other dynamic events within cells have been largely invisible, but will be increasingly observable with new imaging modalities. In particular, lanthanide probes, with the dramatic lowering of background achieved through time-gating can enable new microscopic imaging, if successfully coupled with cell penetration and molecular targeting and recognition. This new scientific capability is certain to have significant commercial appeal and adoption in the basic science and medical research markets.