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

相敏流式细胞术是一种传统的、传播最小的方法，可以捕获每个事件的平均荧光寿命。也就是说，早期的频域细胞术技术涉及到高度的复杂性和笨重的模拟硬件。由于这种技术的复杂性，细胞学界一直缺乏一种广泛而可靠的方法来捕捉激发态衰变。该项目探索了新的数字方法，作为在细胞术中引入多个生命周期的额外分离能力的手段。这些包括：(1)引入多频调制，(2)通过非调制数字信号处理实现捕获时间分辨信号的创新方法，以及(3)将荧光寿命参数应用于分选系统，以便根据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，而且能够测量多指数衰减的细胞计数系统很有吸引力，其原因包括：改善自身荧光与外源信号的分离，捕获流动中表面增强的拉曼散射，以及在细胞分选过程中定量荧光蛋白表达。多寿命细胞仪的发展将对整个细胞术界产生重大影响。分选和分析的时间方法可能直接影响流式细胞术中广泛的染色和分析方案，提高低密度抗原研究的准确性，揭示可能出现的罕见事件，表现出独特的激发态衰减，并允许各种用户在其商业仪器上实现该功能。兴奋状态细胞术技术的进步也将扩大新墨西哥州立大学（一所西班牙裔服务机构）中代表性不足的本科生和研究生的参与。从事细胞仪开发的少数人将接触到高影响力的生物工程、化学工程和项目成果所需的其他多学科领域。所有或部分描述的细胞术技术的最终目标是融入生物技术产业。所有项目成果都可以在主要研究者研究网站上阅读：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.