FLOW CYTOMETER FOR QUANTIFYING FLUORESCENCE LIFETIME

流式细胞术测定荧光寿命的技术 (FCM)用荧光染料标记的单细胞的研究正在进行中。 远期目标是发展FCM技术， 来自与细胞内大分子成分结合的荧光染料的信号 有重叠的发射光谱，但不同的寿命， 量化荧光寿命作为研究荧光染料的参数 与细胞成分和彼此之间的相互作用。 具体 本项目的目标是：（1）评估第一代仪器 在灵敏度、精度、动态范围和精度方面， 寿命测量;（2）确定仪器的能力， 分辨来自光谱重叠荧光染料的发射信号， 包括抑制背景干扰的有效性， 通过细胞自发荧光，非特异性染色，游离和未结合染料， 和拉曼散射;（3）将该技术作为光谱工具 用于探测荧光染料与细胞和染色体的相互作用; （4）随着用户需求的增加， 生物医学社区 当染色的细胞流过 腔室并与强度调制的激光束相交。 脉搏 调制的荧光信号，其在相位上从 由相敏检测（PSD）电子器件处理 基于寿命抑制多组分混合物中的一种组分 差或确定相移和解调程度 其允许计算荧光寿命。 流动系统， 包括激光调制器、改进的流动室、荧光检测器 信号放大器和相敏检测器是可操作的， 建立了信号检测理论模型。 初步 实验已经证明（1）荧光检测阈值 （灵敏度）300至800个异硫氰酸荧光素（FITC）分子 激励频率的相敏测量等效 频率范围为10 ~ 30 MHz，（2）变异系数小于1.5% 对排列荧光微球的影响小于4%，对碘化丙啶的影响小于4% 在10 MHz下激发的（PI）染色（DNA含量）CHO细胞系，和（3） 能够通过荧光相位量化纳秒寿命， 溴化乙锭、PI和FITC染色细胞的振幅测量。 来自用Pi和FITC组合染色的细胞的荧光信号 也已解决，并显示基于 寿命差异（相移）。 我们将继续评估 第一代仪器在仪器能力方面， 作为探测荧光染料与 细胞和染色体。 染色体结构、DNA合成的研究 与细胞周期的关系，抗体标记的异质性，以及 膜受体结构的计划。 提出了改进建议， 根据需要开发更先进和多功能的仪器， 生物群落

The technology for measuring fluorescence lifetime by flow cytometry (FCM) in single cells labeled with fluorochromes is under development. The long-term goals are to develop the FCM technology for resolving signals from fluorescent dyes bound to macromolecular components In cells that have overlapping emission spectra, but different lifetimes and to quantify fluorescence lifetime as a parameter for studying fluorochrome interactions with cellular constituents and each other. The specific aims of this project are: (1) to evaluate the first generation instrument in terms of sensitivity, precision, dynamic range, and accuracy of lifetime measurements; (2) to determine the instrument's capability to resolve the emission signals from spectrally-overlapping fluorochromes, including the effectiveness in suppressing background interference caused by cellular autofluorescence, nonspecific staining, free and unbound dye, and Raman scatter; (3) to apply the technology as a spectroscopic tool for probing the interactions of fluorochromes with cells and chromosomes; and (4) to improve on the technology as user needs arise from the biomedical community. Stained cells are analyzed as they flow through a chamber and intersect an intensity-modulated laser beam. The pulse-- modulated fluorescence signals, which are shifted in phase from a reference, are processed by phase-sensitive detection (PSD) electronics to suppress one component in a multicomponent mixture based on lifetime differences or to determine the phase shift and extent of demodulation which allows calculation of fluorescence lifetimes. The flow system, including laser modulator, improved flow chamber, fluorescence detector, signal amplifiers, and phase-sensitive detectors, is operational and a model of the signal detection theory has been developed. Preliminary experiments have demonstrated (1) a fluorescence detection threshold (sensitivity) of 300 to 800 fluorescein isothiocyanate (FITC) molecules equivalence by phase sensitive measurement for excitation frequencies ranging from 10 to 30 MHz, (2) coefficients of variation less than 1.5% on alignment fluorospheres and less than 4% on propidium iodide (PI)-stained (DNA content) line CHO cells excited at 10 MHz, and (3) the capability to quantify nanosecond lifetimes by fluorescence phase and amplitude measurements on ethidium bromide-, PI-, and FITC-stained cells. Fluorescence signals from cells stained with Pi and FITC in combination also have been resolved and the individual histograms displayed based on differences in lifetimes (phase shift). We will continue to evaluate the first generation instrument in terms of instrumental capabilities and its usefulness as a tool for probing the interactions of fluorochromes with cells and chromosomes. Studies of chromatin structure, DNA synthesis in relationship to cell cycle, heterogeneity of antibody labeling, and membrane receptor structure are planned. Improvements are proposed to develop a more advanced and versatile instrument as needs arise from the biological community.