Number and brightness analysis (N&B) is a useful tool for the simultaneous visualization of protein oligomers and their localization, with single-molecule sensitivity. N&B determines particle brightness (fluorescence intensity per particle) and maps the spatial distribution of fluorescently labeled proteins by performing statistical analyses of the image series obtained using laser scanning microscopy. The brightness map reveals presence of the oligomers of the targeted protein and their distribution in living cells. However, even when corrections are applied, conventional N&B is affected by afterpulsing, shot noise, thermal noise, dead time, and overestimation of particle brightness when the concentration of the fluorescent particles changes during measurement. The drawbacks of conventional N&B can be circumvented by using two detectors, a novel approach that we henceforth call two-detector number and brightness analysis (TD-N&B), and introducing a linear regression of fluorescence intensity. This statistically eliminates the effect of noise from the detectors, and ensures that the correct particle brightness is obtained. Our method was theoretically assessed by numerical simulations and experimentally validated using a dilution series of purified enhanced green fluorescent protein (EGFP), EGFP tandem oligomers in cell lysate, and EGFP tandem oligomers in living cells. Furthermore, this method was used to characterize the complex process of ligand-induced glucocorticoid receptor dimerization and their translocation to the cell nucleus in live cells. Our method can be applied to other oligomer-forming proteins in cell signaling, or to aggregations of proteins such as those that cause neurodegenerative diseases.

中文翻译：

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双检测器数量和亮度分析揭示活细胞中蛋白质的时空寡聚化

数量和亮度分析 (N&B) 是一种有用的工具，可用于同时可视化蛋白质寡聚体及其定位，具有单分子灵敏度。N&B 通过对使用激光扫描显微镜获得的图像系列进行统计分析，确定粒子亮度（每个粒子的荧光强度）并映射荧光标记蛋白质的空间分布。亮度图揭示了目标蛋白质寡聚体的存在及其在活细胞中的分布。然而，即使应用了校正，传统的 N&B 也会受到后脉冲、散粒噪声、热噪声、死区时间以及在测量过程中荧光颗粒浓度发生变化时对颗粒亮度的高估的影响。传统N&的缺点 B 可以通过使用两个检测器来规避，这种新方法我们以后称之为双检测器数量和亮度分析 (TD-N&B)，并引入了荧光强度的线性回归。这在统计上消除了探测器噪声的影响，并确保获得正确的粒子亮度。我们的方法通过数值模拟进行了理论上的评估，并使用一系列稀释的纯化增强型绿色荧光蛋白 (EGFP)、细胞裂解物中的 EGFP 串联寡聚体和活细胞中的 EGFP 串联寡聚体进行了实验验证。此外，该方法用于表征配体诱导的糖皮质激素受体二聚化的复杂过程及其向活细胞中细胞核的易位。我们的方法可以应用于细胞信号传导中的其他寡聚体形成蛋白，