Analysis of fluorescence decay kinetics from variable-frequency phase shift and modulation data.
Analysis of fluorescence decay kinetics from variable-frequency phase shift and modulation data.
复制标题
根据变频相移和调制数据分析荧光衰减动力学。
DOI：
10.1016/s0006-3495(84)84043-6
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发表时间：
1984
影响因子：
3.4
通讯作者：
M. Limkeman
中科院分区：
文献类型：
--
作者：
J. Lakowicz;G. Laczkó;H. Cherek;E. Gratton;M. Limkeman
最近，它已成为可能的测量荧光相移和调制数据在很宽的范围内的调制频率。在本文中，我们描述了这些数据的非线性最小二乘法的分析，以确定指数衰减荧光团的混合物的寿命和分数强度的值。分析模拟数据使我们能够确定那些对于成功解决荧光团混合物发射最关键的实验因素。最关键的因素是实验数据的准确性，个体衰变时间的相对差异，以及在多个发射波长下测量的数据的包含。在八个宽间隔的调制频率下进行测量后，额外的测量仅产生分辨率的适度增加。特别是，在参数的不确定性降低约为调制频率的数量的平方根的倒数。我们的模拟表明，目前可用的精度和数据的一个发射带通，两个衰减时间可以准确地确定，如果它们的比率大于或等于1.4。三个指数衰变也可以解决，但只有当寿命的范围是五倍或更大。为了可靠地确定紧密间隔的衰减时间，在多个发射波长下测量数据，使得组分的分数强度可以变化。此外，任何参数的独立知识大大提高了其余参数可以确定的准确性。在随后的论文中，我们提出了实验结果，广泛证实了预测的解决潜力的变频相位调制荧光。
Recently it has become possible to measure fluorescence phase-shift and modulation data over a wide range of modulation frequencies. In this paper we describe the analysis of these data by the method of nonlinear least squares to determine the values of the lifetimes and fractional intensities for a mixture of exponentially decaying fluorophores. Analyzing simulated data allowed us to determine those experimental factors that are most critical for successfully resolving the emissions from mixtures of fluorophores. The most critical factors are the accuracy of the experimental data, the relative difference of the individual decay times, and the inclusion of data measured at multiple emission wavelengths. After measuring at eight widely spaced modulation frequencies, additional measurements yielded only a modest increase in resolution. In particular, the uncertainty in the parameters decreased approximately as the reciprocal of the square root of the number of modulation frequencies. Our simulations showed that with presently available precision and data for one emission bandpass, two decay times could be accurately determined if their ratio were greater than or equal to 1.4. Three exponential decays could also be resolved, but only if the range of the lifetimes were fivefold or greater. To reliably determine closely-spaced decay times, the data were measured at multiple emission wavelengths so that the fractional intensities of the components could be varied. Also, independent knowledge of any of the parameters substantially increased the accuracy with which the remaining parameters could be determined. In the subsequent paper we present experimental results that broadly confirm the predicted resolving potential of variable-frequency phase-modulation fluorometry.
影响因子：
2.9
作者：
KNUTSON, JR;WALBRIDGE, DG;BRAND, L
通讯作者：
BRAND, L