CAREER: Fluorescence Lifetime in Our Lifetime: Discovery of Approaches to Measure Molecular Excited State Kinetics and Fluorescence Decay by Flow Cytometry

职业生涯：荧光寿命在我们有生之年：发现用流式细胞仪检测分子激发态动力学和荧光衰减的现代方法几十年来，高通量单细胞计数，发现重要的细胞内功能，以及了解单细胞对外部刺激的反应都已经成为可能，这种设备被称为流式细胞仪。流式细胞仪用于研究以及需要获得从血液中收集的细胞的准确数据的临床环境中，以提供艾滋病毒等疾病的预后。细胞仪的工作原理是使单个细胞内或细胞上的蛋白质和其他分子物种达到更高的能量水平，因为细胞通过流体室并在微秒的传输时间内穿过激光束。尽管细胞检测系统在商业上很流行，但现有的细胞检测设备并不能捕捉到单个细胞的“光动力学”特性。也就是说，目前没有一种细胞仪测量细胞内或细胞上分子的荧光衰减和平均荧光寿命。这种能力很重要，因为依赖时间的信息在细胞定量计数、细胞分选和提高细胞分析的信噪比中非常有价值。缺乏依赖于荧光寿命的细胞术主要是由于测量激发态动力学所需的困难和复杂性；这种缺乏反过来又导致缺乏分析和依赖于荧光衰减的应用。因此，这一职业发展计划涉及到发现依赖时间的流式细胞术的新方法，并引入了将基于荧光动力学的测量轻松集成到商业细胞仪系统中的新方法。此外，这项研究还促进了时间分辨流式细胞术的细胞应用。这项工作的具体目标是(I)提供对细胞内固有蛋白质因细胞周期和细胞活力的差异而发生的自发荧光寿命变化的了解，(Ii)探索与细胞内蛋白质运输、蛋白质-蛋白质相互作用和蛋白质复合体形成相关的荧光蛋白质寿命变化，这些变化可通过Forster共振能量转移(FRET)和FRET的损失来检测；以及(Iii)基于微球结合的纳米颗粒的超快速激发态衰减时间，这些纳米颗粒表现出表面增强拉曼散射用于多重微珠分析。新墨西哥州立大学(NMSU)是许多因经济和文化原因留在该州边界内的未被充分代表的少数族裔(URM)学生的所在地。NMSU生物工程学科的培训机会有限，尽管学生的兴趣很高，而且新墨西哥州生物技术和生物科学产业历史上非常优秀。为了增加NMSU学生以及提供给该州土地授予机构的周围社区的教育机会，NMSU的西班牙裔本科生、研究生、五年级学生和K-8教育工作者将被纳入一项教育计划，该计划(1)实施通过研究学习的活动，(2)培养K-8教育外展。将启动与NMSU的西班牙裔学生以及一所文化边缘化的科学磁石小学的直接互动，以利用流式细胞术的研究活动帮助建立综合科学课程。新墨西哥州在流式细胞术方面有着丰富的历史，它在一定程度上是在那里发明的。这一教育计划包含了向理科学生传达的现实世界的解决方案。它还强调了历史意义，并提供了一个背景，说明他们作为科学学习者是谁，以及他们作为新墨西哥州原住民能够取得什么成就。所有项目成果都可以在首席调查员的研究网站上看到：http://che.nmsu.edu/JPH/index.html.

CAREER: FLUORESCENCE LIFETIME IN OUR LIFETIME: DISCOVERY OF MODERN APPROACHES FOR THE DETECTION OF MOLECULAR EXCITED-STATE KINETICS AND FLUORESCENCE DECAY BY FLOW CYTOMETRYFor several decades, high-throughput single cell counting, discovery of important intracellular functions, and an understanding of single cell responses to external stimuli have been possible with the aid of devices called flow cytometers. Flow cytometers are used for research as well as in clinical settings where there is a need to obtain an accurate account of cells collected from blood to provide prognoses for diseases such as HIV. Cytometers work by causing proteins and other molecular species in or on individual cells to reach elevated energy levels as the cells travel through fluidic chambers and traverse laser beams over microsecond transit times. Despite the prevalence of cytometry systems commercially, available cytometry devices do not capture "photodynamic" properties from individual cells. That is, no current cytometry instrument measures the fluorescence decay and average fluorescence lifetime from molecules in or on cells. This capability is important because time-dependent information is very valuable in quantitative cell counting, cell sorting, and improvement of signal-to-noise among cellular assays. The lack of fluorescence lifetime-dependent cytometry is mainly due to the difficulties and complexities required for measuring excited state kinetics; this dearth has in turn resulted in a lack of assays and fluorescence decay-dependent applications. Thus, this CAREER development plan involves the discovery of new approaches for time-dependent flow cytometry and introduces new ways to easily integrate fluorescence dynamic-based measurements into commercial cytometry systems. Moreover, this research advances cellular applications for time-resolved flow cytometry. Specific objectives of this work are (i) to provide an understanding of autofluorescence lifetime changes of intrinsic cellular proteins with differences in cell cycle and cell viability, (ii) to explore fluorescent protein lifetime changes associated with intracellular protein transport, protein-protein interactions and protein complex formation detectable by Forster resonance energy transfer (FRET) and loss of FRET; and (iii) to identify ultra-rapid excited state decay times based on microsphere-bound nanoparticles that exhibit surface-enhanced Raman scattering for multiplex bead assays.New Mexico State University (NMSU) is home to many underrepresented minority (URM) students who remain within the state's borders for financial and cultural reasons. The training opportunities at NMSU in the discipline of bioengineering are limited despite high student interest and the historical excellence of biotechnology and bioscience industry in New Mexico. In an effort to increase educational opportunities for NMSU students as well as the surrounding communities that feed into the state's Land Grant Institution, Hispanic NMSU undergraduates, graduate students, 5th grade students, and K-8 educators will be enlisted in an educational plan that (1) implements learning-through-research activities and (2) cultivates K-8 educational outreach. Direct interaction with Hispanic students at NMSU as well as a culturally marginalized science-magnet elementary school will be initiated to help build integrated science curricula using research activities in flow cytometry. New Mexico has a rich history in flow cytometry, where it was in part invented. This educational plan incorporates real-world solutions to be conveyed to science students. It also emphasizes the historical significance and provides a context on who they are as science learners and what they, as native New Mexicans, can achieve. All project outcomes can be seen at the Principal Investigator's research website: http://che.nmsu.edu/JPH/index.html.