Measurement of monomer-oligomer distributions via fluorescence moment image analysis
Measurement of monomer-oligomer distributions via fluorescence moment image analysis
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DOI：
10.1529/biophysj.106.091181
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发表时间：
2006-11-01
影响因子：
3.4
通讯作者：
Wiseman, Paul W.
中科院分区：
文献类型：
--
作者：
Sergeev, Mikhail;Costantino, Santiago;Wiseman, Paul W.
我们对单个激光扫描显微镜图像的荧光强度波动进行高阶矩分析，应用于研究单体-低聚物分布。我们证明，可以通过分析特定范围的密度和颗粒亮度比的单个图像的荧光强度波动的高阶矩来确定单体和低聚物混合群体的数密度和亮度比。使用荧光微球和细胞进行计算机模拟和实验，以说明这种统计方法的检测限和准确性。模拟结果表明，二聚体或低聚物群体的浓度应小于或等于单体浓度，该方法才能提供准确的结果，并且单体群体的密度检测上限比低聚物的浓度高一个数量级。我们采用该技术来解析具有不同亮度比的两个荧光微球群，并且我们还应用矩分析方法来检查人成纤维细胞中PDGF-β受体的聚集状态的分布。该方法能够相对于非特异性结合荧光团的背景分布解析 PDGF-β 受体的四聚体群体。
We present higher-order moment analysis of fluorescence intensity fluctuations from individual laser scanning microscopy images applied to study monomer-oligomer distributions. We demonstrate that the number densities and brightness ratios of a mixed population of monomers and oligomers can be determined by analyzing higher-order moments of the fluorescence intensity fluctuations from individual images for specific ranges of densities and particle brightness ratios. Computer simulations and experiments with fluorescent microspheres and cells were performed to illustrate the detection limits and accuracy of this statistical approach. The simulation results show that the concentration of the dimer or oligomer population should be less than or equal to the monomeric concentration for the method to provide accurate results, and that the upper density detection limit of the population of monomers is one order-of-magnitude higher than the concentration of the oligomers. We implemented this technique to resolve two populations of fluorescent microspheres with different brightness ratios and we also applied the moment-analysis method to examine the distribution of aggregation states of PDGF-beta receptors in human fibroblast cells. The method was able to resolve a tetrameric population of the PDGF-beta receptors relative to the background distribution of nonspecifically bound fluorophore.