FLOW CYTOMETER FOR QUANTIFYING FLUORESCENCE LIFETIME

该项目的长期目标是发展先进的 流式细胞术测量相分辨荧光的方法 结合在细胞上的荧光染料的发射和激发态寿命 和染色体。本项目的具体目标是：1)应用 将这项技术推广到广泛的生物系统中 这些独特的测量能力的优势；2)确定 低层探测和测量技术的局限性 背景中的荧光探针的发射信号是由 细胞自发荧光，通过光谱发射重叠在 通过非结合/非特异性荧光团的荧光检测通道 标记和拉曼/瑞利散射；3)改进和推进 一种制备相分辨多色荧光的技术 用单调制或双调制激光测量寿命 激动人心。相敏流式细胞仪项目相对 新的，以至于它的许多潜在的应用还没有完全 探索和发展。然而，因为它可以分离荧光 电子和光学发射，量化寿命(S) 直接作为参数，也使常规的流式细胞术 测量，它有广泛的技术上的可能性 申请。这项新技术将增加 可用于多标记应用的荧光标记， 通过提高测量精度和 灵敏度和减少背景干扰，并通过 生物医学研究，这项技术将显著扩大 研究人员对细胞生物过程的理解， 亚细胞和分子水平。本报告中的一个研究亮点 描述了使用荧光寿命作为一种 当发射光谱为 重叠。

The long-term goal of this project is the development of advanced flow cytometric methods for measuring phase-resolved fluorescence emissions and excited-stated lifetimes on fluorochromes bound to cells and chromosomes. The specific aims of this project are 1) to apply the technology to a wide range of biological systems that take advantage of these unique measurement capabilities; 2) determine the limits of the technology for detecting and measuring low-level emission signals from fluorescent probes in backgrounds caused by cellular autofluorescence, by spectral emission overlap among fluorescence detection channels, by unbound/nonspecific fluorophore labeling, and by Raman/Rayleigh scatter; and 3) to improve and advance the technology for making phase-resolved multicolor fluorescence and lifetime measurements using single- or dual-modulated laser excitation. The phase-sensitive flow cytometer project is relatively new so that many of its potential applicati ons have not been fully explored and developed. However, because it can separate fluorescence emissions both electronically and optically, quantify lifetime(s) directly as a parameter, and also make conventional flow cytometric measurements, it has a wide range of technically possible applications. This new technology will increase the range of fluorescent markers that can be used in multi-labeling applications, yield more accurate results by enhancing measurement precision and sensitivity and reducing background interferences, and through biomedical research, the technology will significantly expand the researchers' understanding of biological processes at the cellular, subcellular, and molecular level. A Research Highlight in this report describes the use of fluorescence lifetime as a means of distinguishing between fluorophors when the emission spectra are overlapping.