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

我们一生中的荧光寿命：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，在流式细胞仪的帮助下，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成为可能。流式细胞仪用于研究以及临床环境，在这些环境中需要获得从血液中收集的细胞的准确记录，以便为艾滋病毒等疾病提供预后。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达到更高的能量水平，当细胞在微秒的传输时间内穿过流体室和激光束时。尽管商业上的细胞计系统很流行，但现有的细胞计设备并不能捕获单个细胞的“光动力学”特性。也就是说，目前没有一种细胞仪可以测量细胞内或细胞外分子的荧光衰减和平均荧光寿命。这种能力很重要，因为时间相关信息在定量细胞计数、细胞分选和改进细胞检测中的信噪比中非常有价值。荧光寿命依赖性细胞术的缺乏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检测和依赖荧光衰减的应用。因此，本职业发展计划涉及发现时间依赖性流式细胞术的新方法，并引入新的方法，轻松地将基于荧光动态的测量集成到商业流式细胞术系统中。此外，该研究还促进了时间分辨流式细胞术在细胞中的应用。这项工作的具体目标是：(i)提供对细胞周期和细胞活力差异下细胞内蛋白质自身荧光寿命变化的理解，（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物形成相关的荧光蛋白寿命变化，可通过福斯特共振能量转移（FRET）和FRET损失检测到；（iii）确定基于微球结合纳米颗粒的超快速激发态衰减时间，这些纳米颗粒在多重头分析中表现出表面增强的拉曼散射。新墨西哥州立大学（NMSU）是许多未被充分代表的少数民族（URM）学生的家园，他们出于经济和文化原因留在该州境内。尽管学生的兴趣很高，而且新墨西哥州的生物技术和生物科学产业历史悠久，但新墨西哥州国立大学生物工程学科的培训机会有限。为了增加新密歇根州立大学学生以及周边社区的教育机会，新密歇根州立大学的西班牙裔本科生、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，该计划将(1)实施通过研究学习的活动，(2)培养K-8教育的推广。将与新密歇根州立大学的西班牙裔学生以及一所文化边缘化的科学磁铁小学进行直接互动，以帮助利用流式细胞术的研究活动建立综合科学课程。新墨西哥州在流式细胞术方面有着丰富的历史，流式细胞术在一定程度上是在那里发明的。这个教育计划将现实世界的解决方案传达给理科生。它还强调了历史意义，并提供了他们作为科学学习者的背景，以及他们作为土生土长的新墨西哥人可以取得的成就。所有项目成果都可以在首席研究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.