Microflow time-resolved cytometry for FRET and fluorescent protein development

总结 此应用程序请求购买荧光寿命成像的补充支持 显微镜（FLIM）。这种类型的新显微镜将大大推进该项目的目标： R 01 GM 129859用于FRET和荧光蛋白开发的微流时间分辨流式细胞术。 FLIM系统具有对细胞成像的独特能力，由此图像是荧光的呈现 以高分辨率跨细胞测量寿命。荧光寿命是平均时间a 荧光团处于激发态;它携带大量关于荧光团的信息 例如微环境、近邻邻近性和细胞内分子的运动。这将 支持父R 01项目，因为这项工作的目标是设计和调整荧光寿命 流式细胞术检测到微芯片平台上，用于细胞筛选和最终分选。目标将开发一部小说 微流式细胞仪，将，当完全审查，包括许多独特的功能，如声学 通过微流体通道聚焦细胞，多频率测量，产生多个- 每个细胞的荧光寿命值，以及基于荧光的最终细胞分选的电光设计 寿命测量同时对微流式细胞仪的设计和构建进行了两大方面的研究 该工具的应用正在计划/进行中。这些是1）Förster共振能量的量化 哺乳动物细胞内的FRET转移事件，以及2）近红外荧光蛋白的富集 基于光动力学数据。因此，具有独特测量荧光能力的显微镜（FLIM） 衰减动力学是必不可少的，原因有四：1）它将使我们能够独立地交叉验证我们的细胞计数法 测量，从而提供分辨率和精度的数量级更大的可靠 优化; 2）它将显着增强我们正在探索与我们的新的协作应用程序 3）它将大大扩展我们的细胞仪在其他领域的适用性， 扩大生物医学的影响，同时支持未来的赠款申请;和4）它将成为一个工具， 培训和发展的学生和合作教师在新墨西哥州州立大学，西班牙裔 服务机构。

Summary This application requests supplemental support for the purchase of a fluorescence lifetime imaging microscope (FLIM). A new microscope of this type will significantly advance the aims of the project: R01GM129859 Microflow time-resolved Flow Cytometry for FRET and Fluorescence Protein Development. FLIM systems have unique abilities to image cells whereby the image is a rendering of the fluorescence lifetimes measured across cells at a high resolution. The fluorescence lifetime is the average time a fluorophore spends in the excited state; it carries a significant amount of information about the fluorophore such as the microenvironment, near-neighbor proximity, and movement of intracellular molecules. This will support the parent R01 project because the objective for this work is to design and adapt fluorescence lifetime flow cytometry onto a microchip platform for cell screening and eventual sorting. The aims will develop a novel microflow cytometer that will, when completely vetted, incorporate many unique features such as acoustic focusing of cells through microfluidic channels, multi-frequency measurements that give rise to multiple- fluorescence lifetime values per cell, and electro-optical design for eventual cell sorting based on fluorescence lifetime measurements. Concurrently with the design and construction of the microflow cytometer two major applications of this tool are planned/underway. These are 1) the quantification of Förster resonance energy transfer (FRET) events inside of mammalian cells, and 2) the enrichment of near-infrared fluorescent proteins based on photo-kinetic data. Therefore a microscope (FLIM) with the unique ability to measure fluorescence decay kinetics is essential for 4 reasons: 1) it will enable us to independently cross-validate our cytometry measurements thereby providing resolution and precision that are orders of magnitude greater for reliable optimization; 2) it will significantly enhance the collaborative applications that we are exploring with our novel cytometer and current aims; 3) it will greatly expand the applicability of our cytometer for other fields thus broadening the biomedical impact while supporting future grant applications; and 4) it will become a tool for training and development of students and collaborating faculty at New Mexico State University, a Hispanic Serving Institution.