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

这一小型企业创新研究(SBIR)第二阶段项目解决了当前在活细胞中蛋白质-蛋白质相互作用的显微成像方法中发现的挑战。这些方法广泛依赖于青色(CFP)和黄色荧光蛋白(YFP)之间的Förster共振能量转移(FRET)。由于背景噪声的干扰和这些荧光团的固有光物理性质，这些方法是有问题的。我们开发了一种使用稀土络合物供体与时间分辨荧光显微镜相结合的系统，它克服了这些限制。本项目的研究目标是扩大这些发光探头的能力，并进行放大，以提供足够的材料来满足初期产品销售的制造需求。到该项目结束时，我们将拥有在活细胞中进行现场定向时间分辨测量的试剂和可操作的时间分辨成像模块的原型。如果该项目成功，该项目的更广泛的影响/商业潜力是开发一类新的细胞成像试剂和技术的潜力。这一创新将提高研究人员追踪蛋白质-蛋白质相互作用途径的能力，其定量准确性是以前所没有的。这不仅将影响基础和应用研究，还将通过发现新的诊断和治疗疾病的药物靶点和机制，影响初级保健。设计和使用新的探针来研究活细胞在分子和亚细胞水平上的结构和功能是一个非常重要的课题，越来越需要新的方法和工具来可视化分子物种在细胞中的分布，以及它们相互作用的方式。蛋白质-蛋白质相互作用和细胞内的其他动态事件在很大程度上是看不见的，但随着新的成像模式的出现，这些事件将越来越明显。特别是，通过时间门控实现的本底显著降低的镧系元素探针，如果成功地与细胞渗透、分子靶向和识别相结合，就可以实现新的显微成像。这种新的科学能力肯定会在基础科学和医学研究市场上具有巨大的商业吸引力和采用率。

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 complex donor 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 product sales. 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 of cell 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 through the 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.