IDBR: Development of Heterogeneous Excited State Flow Cytometry Sorting and Analysis

相敏流式细胞术是一种传统的和最小传播的方法，在逐个事件的基础上捕获平均荧光寿命。 也就是说，频域细胞计数技术的早期体现涉及具有笨重的模拟硬件的高度复杂性。 由于这些技术的复杂性，细胞组学界缺乏广泛和强大的手段来捕获激发态衰变。 该项目探索了新的数字方法，作为引入多个寿命的手段，以提高细胞仪的分离能力。 其中包括：（1）多频调制的引入，（2）通过非调制数字信号处理捕获时间分辨信号的创新方法的实施，和（3）将荧光寿命参数应用于分选系统，以便基于衰变动力学测量分离细胞或颗粒。一种不仅能够测量平均荧光寿命而且能够进行多指数衰减的细胞计数系统是有吸引力的，原因有几个，包括：改善自体荧光与外源信号的分离，捕获流动中的表面增强拉曼散射，以及在细胞分选期间定量荧光蛋白表达。 多寿命细胞计数仪器的开发将对整个细胞计数界产生重大影响。 用于分选和分析的时间方法可以直接影响流式细胞术中的广泛的染色和分析方案，提高低密度抗原研究的准确性，揭示表现出独特的激发态衰变的可能的罕见事件，并允许各种用户在其商业仪器上实现该能力。 兴奋状态细胞计数技术的进步也将扩大参与不足的本科生和研究生在新墨西哥州州立大学，西班牙裔服务机构。 从事细胞仪开发的少数民族将接触到项目成果所需的高影响力生物工程，化学工程和其他多学科领域。 所有或部分所述细胞计数技术的最终目标是整合到生物技术行业中。 所有项目成果都可以在主要研究者研究网站上查阅：http://che.nmsu.edu/JPH/index.html。

Phase-sensitive flow cytometry is a traditional and minimally disseminated method of capturing the average fluorescence lifetime on an event-by-event basis. That is, early incarnations of frequency-domain cytometry techniques involved a high degree of complexity with cumbersome analog hardware. Owing to the intricate nature of such techniques, the cytomics community has lacked a widespread and robust means of capturing excited-state decays. This project explores new digital methods as a means to introduce multiple lifetimes for additional separation power in cytometry. These include: (1) introduction of multiple-frequency modulation, (2) implementation of innovative approaches for capturing time-resolved signals through non-modulated digital signal processing, and (3) application of fluorescence lifetime parameters to sorting systems so as to separate cells or particles based on decay kinetic measurements. A cytometry system capable of not only average fluorescence lifetime measurements but also multi-exponential decay is appealing for several reasons which include: improving the separation of autofluorescence from exogenous signals, capturing surface enhanced Raman scattering in flow, and quantifying fluorescence protein expression during cell sorting. The development of a multiple lifetime cytometry instrument will significantly impact the cytometry community at large. A temporal method for sorting and analysis may directly affect a wide range of staining and analysis protocols in flow cytometry, improve accuracy of low-density antigen studies, reveal possible rare events that exhibit unique excited state decays, and allow users of all kinds to implement the capability on their commercial instrument. The advancement of excited-state cytometry techniques will also broaden the participation of underrepresented undergraduate and graduate students at New Mexico State University, a Hispanic Serving Institution. Minorities working on the development of the cytometer will be exposed to high-impact bioengineering, chemical engineering, and other multidisciplinary fields needed for the project outcomes. The end goal for all or part of the described cytometry techniques is the integration into the biotechnology industry. All project outcomes can be perused at the Principle Investigator research website: http://che.nmsu.edu/JPH/index.html.