FLOW CYTOMETER FOR QUANTIFYING FLUORESCENCE LIFETIME

用流式细胞术测量荧光寿命的技术 用荧光标记的单细胞(FCM)正在开发中。 我们的长期目标是开发FCM技术来解决 来自荧光染料的信号与细胞中的大分子成分结合 有重叠的发射光谱，但寿命不同， 荧光寿命的量化作为研究荧光色素的参数 与细胞成分以及彼此之间的相互作用。具体的 本项目的目标是：(1)对第一代仪器进行评估 在灵敏度、精确度、动态范围和精确度方面 寿命测量；(2)确定仪器的能力 解析来自光谱重叠的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，自由和非结合染料， 和拉曼散射；(3)将该技术作为光谱工具应用 用于探索荧光染料与细胞和染色体的相互作用； 以及(4)根据用户的需求改进技术 生物医学社区。染色的细胞在流经 并与强度调制的激光光束相交。脉搏-- 调制的荧光信号，它从一个 参考，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个组分 差还是要确定相移和解调的程度 这允许计算荧光寿命。流动系统， 包括激光调制器、改进的流动室、荧光检测器、 信号放大器和相敏探测器已投入使用， 建立了信号检测理论模型。初步 实验证明：(1)荧光检测阈值 300~800个异硫氰酸荧光素(FITC)分子的(灵敏度) 相敏测量励磁频率的等效性 10至30兆赫；(2)变异系数小于1.5% 关于氟化球和小于4%的碘化丙啶的排列 (PI)染色(DNA含量)的CHO细胞在10 MHz激发，和(3) 能够通过荧光相位来量化纳秒寿命 溴化乙锭、PI和FITC染色细胞的幅度测量。 PI和FITC联合染色细胞的荧光信号 也进行了解析，并根据 寿命差异(相移)。我们将继续评估 第一代乐器的仪器性能和它的 作为一种工具来探索荧光染料与 细胞和染色体。大鼠染色质结构、DNA合成的研究 与细胞周期的关系、抗体标记的异质性以及 膜受体结构已规划好。建议进行改进，以 开发更先进、更多功能的仪器，以满足 生物圈。

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.