Deka C, Cram L S, Habbersett R, Martin J C, Sklar L A, Steinkamp J A
Group LS-1, MS M888, Los Alamos National Laboratory, New Mexico 87545, USA.
Cytometry. 1995 Dec 1;21(4):318-28. doi: 10.1002/cyto.990210403.
In frequency-domain lifetime spectroscopy, the apparent fluorescence lifetimes obtained from phase-shift measurements are independent of modulation frequency only in the special case of a single exponential fluorescence decay. For heterogeneous fluorescence decay, the apparent fluorescence lifetimes measured by the phase-shift methods are functions of the modulation frequency. This modulation-frequency dependent property of apparent fluorescence lifetimes may be used to identify heterogeneous fluorescence decays by measuring lifetimes at multiple frequencies. In this article we explore the requirements and experimental design considerations for making such measurements in flow. We report a phase-sensitive flow cytometric method that allows one to probe the excited state-lifetimes of labeled cells by using multiple simultaneous modulation frequencies. Application of this method is demonstrated by measuring fluorescence lifetimes of labeled cells at two frequencies simultaneously, using a continuous-wave, dual-frequency modulated excitation in flow. The dual-frequency method presented herein can be used to rapidly identify heterogeneity in the fluorescence decay on a cell-by-cell basis in real time. Information on the nature of the fluorescence decay is important in biological measurements because it can provide insight into intermolecular interactions at the subcellular level.
在频域寿命光谱学中，仅在单指数荧光衰减的特殊情况下，通过相移测量获得的表观荧光寿命才与调制频率无关。对于非均匀荧光衰减，用相移方法测量的表观荧光寿命是调制频率的函数。表观荧光寿命的这种与调制频率相关的特性可用于通过在多个频率下测量寿命来识别非均匀荧光衰减。在本文中，我们探讨了在流动状态下进行此类测量的要求和实验设计考量。我们报告了一种相敏流式细胞术方法，该方法允许通过使用多个同时的调制频率来探测标记细胞的激发态寿命。通过在流动中使用连续波、双频调制激发同时在两个频率下测量标记细胞的荧光寿命，证明了该方法的应用。本文提出的双频方法可用于实时逐个细胞地快速识别荧光衰减的异质性。关于荧光衰减性质的信息在生物学测量中很重要，因为它可以提供对亚细胞水平分子间相互作用的深入了解。
