Phillips G R, Georghiou S
Department of Physics, University of Tennessee, Knoxville 37996-1200.
Biophys J. 1993 Aug;65(2):918-26. doi: 10.1016/S0006-3495(93)81127-5.
Global analysis using trilinear curve resolution is described and shown to be a powerful method for the resolution of polarized fluorescence data arrays, in which the measured fluorescence intensity is a separable function of polarization orientation, excitation wavelength, and emission wavelength. This methodology is applicable to mixtures the components of which have linearly independent excitation and emission spectra and distinct anisotropies. Normalized excitation and emission spectra of individual components can be uniquely determined without prior assumptions concerning spectral shapes (e.g., sum of Gaussians) and without the uncertainties inherent in bilinear techniques such as principal component analysis or factor analysis. The normalized excitation and emission vectors are combined with the total absorption spectrum of the multicomponent mixture to compute absolute absorption and emission spectra. The precision of this methodology is evaluated as a function of noise, overlap, relative intensity, and anisotropy difference between components using simulated mixtures of the DNA bases. The ability of this method to extract individual spectra from steady-state fluorescence data arrays is illustrated for mixtures containing two and three components.
描述了使用三线曲线分辨的全局分析方法，该方法被证明是解析偏振荧光数据阵列的有力方法，其中测量的荧光强度是偏振方向、激发波长和发射波长的可分离函数。该方法适用于其组分具有线性独立激发和发射光谱以及不同各向异性的混合物。无需对光谱形状（例如高斯函数之和）进行先验假设，也无需像主成分分析或因子分析等双线性技术中固有的不确定性，就可以唯一确定各个组分的归一化激发光谱和发射光谱。将归一化的激发矢量和发射矢量与多组分混合物的总吸收光谱相结合，以计算绝对吸收光谱和发射光谱。使用DNA碱基的模拟混合物，将该方法的精度评估为噪声、重叠、相对强度以及组分之间各向异性差异的函数。对于含有两种和三种组分的混合物，展示了该方法从稳态荧光数据阵列中提取各个光谱的能力。
