Fast and Accurate Tools for Measuring Fluorescence in Living Cells

描述（由申请人提供）： 药物发现关键取决于可靠的生物活性测定。活细胞测定为测量生物活性提供了丰富的环境。与遗传编码的荧光生物传感器相结合，活细胞检测有可能为特定信号通路提供前所未有的特异性读数。尽管基因编码荧光生物传感器广泛用于活细胞中的基础研究应用，但由于难以测量和解释荧光强度读数，包括信噪比差，细胞表达的可变性以及化合物发射的荧光的干扰，因此对药物发现的影响很小。该第一阶段项目将展示一种新策略的可行性，该策略将高度特异性的生物传感器与极快的荧光寿命测量相结合，以产生高通量筛选应用所需的速度，灵敏度和特异性。该方法采用基于荧光寿命的替代荧光测量，其比时间相关单光子计数（TCSPC）快得多，但也更精确。它在非成像模式下工作，这使得数据解释简单，并最大限度地减少背景荧光。它远远超出了对基于图像的技术的预期增量改进。我们的初步数据表明，强大的活细胞检测的巨大潜力时，生命周期的方法应用于测量基因编码的荧光传感器。我们的具体目标将完成重要的原则步骤证明，并为我们在5年内建立一个强大的活细胞药物发现平台的长期目标设定方向。 公共卫生相关性：迫切需要新的生物活性检测方法来开发安全有效的药物，以提供更好的治疗结果并改善人类健康。该提案解决了与使用荧光活细胞测定相关的技术挑战，并具有降低成本和提高药物发现过程可靠性的强大潜力。

DESCRIPTION (provided by applicant): Project Summary/Abstract Drug discovery depends crucially upon reliable assays for biological activity. Live cell assays provide a rich environment for measuring biological activity. Coupled with genetically encoded fluorescent biosensors, live cell assays have the potential to provide read-outs with unprecedented specificity for particular signaling pathways. Although widely used for basic research applications in living cells, genetically encoded fluorescent biosensors have had little impact on drug discovery because of difficulties in measuring and interpreting fluorescence intensity read-outs, including poor signal to noise ratios, variability in cell expression, and interference from fluorescence emitted by compounds. This Phase 1 project will demonstrate the feasibility of a new strategy that combines highly specific biosensors with extremely fast fluorescence lifetime measurements to produce the speed, sensitivity and specificity needed for high throughput screening applications. This approach employs an alternative fluorescence measurement based on fluorescence lifetime that is much faster than time-correlated single photon counting (TCSPC), yet also more precise. It operates in non-imaging mode which makes for simple data interpretation and minimizes background fluorescence. It goes far beyond the expected incremental improvements to image-based technologies. Our preliminary data demonstrates the tremendous potential for robust live cell assays when lifetime methodology is applied to measuring genetically encoded fluorescent sensors. Our specific aims will accomplish the vital proof of principle steps and set the direction for our long term objectives of producing a robust live cell drug discovery platform within 5 years. PUBLIC HEALTH RELEVANCE: New assays for biological activity are urgently needed to develop safe and effective drugs that provide better treatment outcomes and improved human health. This proposal addresses the technical challenges associated with using fluorescent live-cell assays and has strong potential to reduce the cost and improve the reliability of drug discovery processes.