We present a fluorescence fluctuation image correlation analysis method that can rapidly and simultaneously measure the diffusion coefficient, photoblinking rates, and fraction of diffusing particles of fluorescent molecules in cells. Unlike other image correlation techniques, we demonstrated that our method could be applied irrespective of a non-uniformly distributed, immobile blinking fluorophore population. This allows us to measure blinking and transport dynamics in complex cell morphologies, a benefit for a range of super-resolution fluorescence imaging approaches that rely on probe emission blinking. Furthermore, we showed that our technique could be applied without directly accounting for photobleaching. We successfully employed our technique on several simulations with realistic EMCCD noise and photobleaching models, as well as on Dronpa-C12 labeled beta-actin in living NIH/3T3 and HeLa cells. We found that the diffusion coefficients measured using our method were consistent with previous literature values. We further found that photoblinking rates measured in the live HeLa cells varied as expected with changing excitation power.

中文翻译：

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复杂细胞空间中蛋白质扩散、探针闪烁和光漂白动力学的快速集合测量

我们提出了一种荧光波动图像相关分析方法，可以快速同时测量细胞中荧光分子的扩散系数、光闪烁率和扩散粒子的分数。与其他图像相关技术不同，我们证明了我们的方法可以应用于不均匀分布的、不动的闪烁荧光团。这使我们能够测量复杂细胞形态中的闪烁和传输动力学，这对依赖探针发射闪烁的一系列超分辨率荧光成像方法是有益的。此外，我们表明我们的技术可以在不直接考虑光漂白的情况下应用。我们成功地将我们的技术应用于具有真实 EMCCD 噪声和光漂白模型的多个模拟，以及在活的 NIH/3T3 和 HeLa 细胞中 Dronpa-C12 标记的β-肌动蛋白。我们发现使用我们的方法测量的扩散系数与以前的文献值一致。我们进一步发现，在活 HeLa 细胞中测得的光闪烁率随激发功率的变化而变化。