Heterogeneous excited state sorting and analysis cytometry

描述（由申请人提供）：这项R15区域申请的重点是开发一种新的生物过程传感技术，旨在加强新墨西哥州立大学化学工程系的生物医学研究和培训。一种新的诊断系统正在开发中，用于快速发展的时间敏感流式细胞术技术。也就是说，提出了新的方法来测量非均质激发态荧光衰减，近瞬时非弹性散射率，荧光寿命，和相位滤波方法从细胞和粒子在瞬态。提出的时间分辨技术进行了实时细胞分析和分选的研究。此外，这些新技术的发展是为了从单个细胞和/或颗粒中识别罕见的衰变动力学现象，探索新兴的细胞术应用，以及研究如何改进和解决常见的细胞术空白，如自体荧光噪声困扰测定。为了支持异质激发态分选和分析细胞术（HESAC）的发展，我们将检验两个假设：(1)内源性细胞发射的高通量多指数荧光衰减测量不仅可以将大量自身荧光从弱外源性发射中分离出来，还可以揭示离散的内在物种，揭示与不同细胞周期和状态下存在的内在蛋白质相关的罕见事件，允许分离拉曼散射进行多路复用，并表明能量转移效应；(2)根据热诱导构象变化（即展开）和振动破坏（即时环境变化）推测具有不同异质寿命的稳定荧光蛋白的直接选择和筛选将受益于基于荧光寿命的分选。在此应用中研究的技术发展和研究问题对更大的细胞术社区具有广泛影响的主要潜力。由于技术特点，如(i)紧凑性，用于即时诊断，（ii）多面性，便于商业整合，（iii）可扩展性，用于发展为生物医学分子成像设备和系统，预计将对细胞术研究做出重大贡献；（iv）复杂，用于探索新兴的细胞术分析和从未研究过的现象。预计高影响细胞术研究的研究将进一步发展到与细胞和颗粒的分选和分析有关的问题。最终，最重要的贡献是新墨西哥州立大学的本科生和研究生接触到新的生物医学工程概念、方法和原理，这些概念、方法和原理与生物学、化学和物理学科的发现相结合，并导致定量异质激发态衰变分析和细胞计数的商业化引入。

DESCRIPTION (provided by applicant): This R15 AREA application focuses on the development of a new bioprocess sensing technology poised to enhance biomedical research and training within the New Mexico State University department of chemical engineering. A new diagnostic system is under development for the rapid advancement of temporally-sensitive flow cytometry techniques. That is, new methodologies are proposed for the measurement of heterogeneous excited state fluorescence decays, near- instantaneous inelastic scattering rates, fluorescence lifetime, and phase-filtering approaches from cells and particles in transient states. The proposed time resolved techniques are investigated for real-time cytometric analysis as well as sorting. In addition, these new technologies are developed to identify rare decay kinetic phenomena from individual cells and/or particles, to explore emerging cytometry applications, and for investigation of was to improve and address common cytometry gaps such as autofluorescence noise-plagued assays. To support the development of heterogenous excited-state sorting and analysis cytometry (HESAC), two hypotheses will be examined: (1) that high-throughput multi-exponential fluorescence decay measurements of endogenous cellular emission will not only separate bulk autofluorescence from weak exogenous emissions but also reveal discrete intrinsic species, expose rare events related to intrinsic proteins present at different cell cycles and states, permit separation of Raman scatter for multiplexing, and indicate energy transfer effects; and (2) that the direct selection and screening of stable fluorescent proteins, which are surmised to have distinct heterogeneous lifetimes based on thermally induced conformational changes (i.e. unfolding) and vibrational disruptions (immediate environmental changes), will benefit from fluorescence lifetime-based sorting. The technology development and research questions to be studied in this application have a major potential for widespread influence on the larger cytometry community. Significant contributions to cytometry research are anticipated owing to technological characteristics such as (i) compactness, for point-of-care diagnostics, (ii) multifaceted, for facile commercial integration, (iii) expandable, for development into biomedical molecular imaging devices and systems; and (iv) sophisticated, for the exploration of emerging cytometry assays and never-before studied phenomena. It is projected that the study of high-impact cytometry research will progress into further questions related to sorting, and analysis of cells and particles. Ultimately the most important contribution is that New Mexico State University undergraduate and graduate students be exposed to novel biomedical engineering concepts, methodologies, and principles that coalesce with discoveries in biology, chemistry, and physics disciplines, and that lead to the commercial introduction of quantitative heterogeneous excited-state decay analysis and sorting cytometry. PUBLIC HEALTH RELEVANCE: Development and validation of heterogeneous excited-state-dependent sorting and analysis cytometry (HESAC) for biomedical research applications, clinical diagnostics, and use in point-of-care cytometry systems.