Microflow time-resolved cytometry for FRET and fluorescent protein development

项目摘要 本研究项目的目标是设计和应用基于荧光衰减动力学的流式细胞术 微芯片平台。该系统将用于量化Förster共振能量转移(FRET)事件 在哺乳动物细胞内，根据它们的光动力学，可以充分丰富近红外荧光蛋白。这个 微流装置将结合独特的功能，如通过微流控对细胞进行声聚焦 通道、产生每个单元的多个荧光寿命值的多频率测量， 捕获多像素荧光寿命测量的成像功能，以及分类功能 依赖于衰变动力学参数。我们的第一个目标将是使用细胞仪对细胞进行计数 荧光(FRET)供体荧光寿命的变化。当FRET由 能量转移荧光团对的激发态动力学变化，结果是一个数据集没有 受到了基于强度的伪影的影响。此外，新的计算工具箱包括基于相量的 FRET轨迹和FRET效率，为细胞筛选开发了细胞学参数，提供了 细胞内寿命的异质性，以每秒数千个细胞的速度。我们在以下地址进行了测试： 细胞表面以及细胞内的FRET生物探针。这两个系统都具有生物医学意义。 与其在筛选过程中的靶标蛋白功能变化有关。这个项目的第二个目标是 采用基于微芯片的系统，并使用它来主动筛选细菌库并对 表达高量子产率的近红外荧光蛋白。量子产额是一种光物理特性 与平均荧光寿命成正比的荧光分子，或平均时间 荧光团处于激发状态。因此，可以基于荧光来分离样品的工具 寿命是相当有价值的，因为平均强度可能会受到其他因素的困扰，例如注意力， 量子效率和仪器文物。我们第二个目标的长期意义是有能力 加快开发用于分子和漫反射光学的近红外荧光蛋白 体层摄影术。一般说来，紧凑、灵敏和依赖时间的细胞学系统的开发是 其影响超出了提出的两项生物医学应用。因此，这项工作是迈向 评估好处，展示数量性质，并为在许多方面广泛使用奠定基础 更多的细胞学应用。

Project Summary The goal of this research project is to design and apply fluorescence decay kinetic-based flow cytometry on a microchip platform. The system will be used to quantify Förster resonance energy transfer (FRET) events inside of mammalian cells and fully enrich near-infrared fluorescent proteins based on their photo-kinetics. The microflow device will incorporate unique features such as acoustic focusing of cells through microfluidic channels, multi-frequency measurements that give rise to multiple-fluorescence lifetime values per cell, imaging capabilities to capture multi-pixel fluorescence lifetime measurements, and sorting capabilities dependent on decay-kinetic parameters. Our first aim will be to use the cytometer to count cells based on changes in the fluorescence (FRET) donor’s changing fluorescence lifetime. When FRET is evaluated by the excited state kinetic changes of the energy-transferring fluorophore pairs, the result is a data set that has not been affected by intensity-based artifacts. Moreover, with new computational toolboxes including phasor-based FRET trajectories and FRET efficiency, cytometric parameters are developed for cell screening that provide heterogeneity of lifetimes within the cell at a rate of thousands of cells per second. We test this with FRET at the cell surface as well as with an intracellular FRET bioprobe. Both systems have biomedical significance related to protein function alteration thereof with targets during screening. The second aim for this project is to take the microchip-based system and use it to actively screen bacterial libraries and sort single cells that express near-infrared fluorescent proteins with high quantum yield. The quantum yield is a photophysical trait of fluorescent molecules that is directly proportional to the average fluorescence lifetime, or average time the fluorophore spends in the excited state. Therefore a tool that can isolate samples based on the fluorescence lifetime is quite valuable since the average intensity can be plagued by other factors such as concentration, quantum efficiency, and instrument artifacts. The long term significance of our second aim is the ability to expedite the development of near-infrared fluorescent proteins for use in molecular and diffuse optical tomography. In general, the development of a compact, sensitive, and time-dependent cytometry system is impacting beyond the two biomedical applications proposed. Accordingly this work is the first step toward evaluating the benefits, demonstrating the quantitative nature, and setting the stage for broad use across many more cytometric applications.