Microflow time-resolved cytometry for FRET and fluorescent protein development

项目摘要 本研究的目的是设计和应用基于荧光衰减动力学的流式细胞仪， 微芯片平台该系统将用于量化福斯特共振能量转移（FRET）事件 在哺乳动物细胞内，并基于其光动力学充分富集近红外荧光蛋白。的 微流装置将结合独特的特征， 通道，多频率测量，产生多个荧光寿命值每细胞， 成像能力，以捕获多像素荧光寿命测量，以及分选能力 取决于衰变动力学参数。我们的第一个目标将是使用细胞计数器计数细胞的基础上 荧光（FRET）供体的荧光寿命的变化。当FRET由 能量转移荧光团对的激发态动力学变化，结果是没有 受到基于强度的伪影的影响。此外，随着新的计算工具箱，包括基于相量的 FRET轨迹和FRET效率、细胞计数参数被开发用于细胞筛选，其提供 细胞内的生命周期的异质性以每秒数千个细胞的速度。我们用FRET测试这个， 细胞表面以及细胞内FRET生物探针。这两个系统都具有生物医学意义 与筛选过程中靶蛋白功能改变有关。该项目的第二个目标是 利用基于微芯片的系统，并使用它来主动筛选细菌文库， 表达高量子产率的近红外荧光蛋白。量子产率是一个物理特性 荧光分子的平均荧光寿命，或平均时间成正比， 荧光团处于激发态。因此，一种可以基于荧光分离样品的工具 寿命是相当有价值的因为平均强度可能受到其它因素如浓度的影响， 量子效率和仪器伪像我们第二个目标的长期意义在于， 加快近红外荧光蛋白的发展，用于分子和扩散光学 断层扫描一般来说，开发紧凑、灵敏和时间依赖性的细胞计数系统是必要的。 影响超出了所提出的两种生物医学应用。因此，这项工作是朝着 评估效益，展示定量性质，并为在许多领域的广泛使用奠定基础。 更多的细胞计数应用。

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.