Combining stimulated emission depletion and fluorescence correlation spectroscopy (STED-FCS) provides a powerful and sensitive tool for studying the molecular dynamics in live cells with high spatio-temporal resolution. STED-FCS gives access to molecular diffusion characteristic at the nanoscale occurring within short period of times. However due to the incomplete suppression of fluorescence in the STED process, the STED-FCS point spread function (PSF) deviates from a Gaussian shape and challenges the analysis of the auto-correlation curves obtained by FCS. Here, we model the effect of the incomplete fluorescence suppression in STED-FCS experiments and propose a new fitting model improving the accuracy of the diffusion times and average molecule numbers measurements. The implementation of a STED module with pulsed laser source on a commercial confocal/FCS microscope allowed us to apply the STED-background corrected model to fit the STED-FCS measurements. The experimental results are in good accordance with the theoretical analysis both for the number of molecules and the diffusion time which decrease accordingly with the STED power.

中文翻译：

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用于无偏 STED-FCS 分析的简单 STED 背景校正拟合模型

结合受激发射损耗和荧光相关光谱 (STED-FCS) 为研究具有高时空分辨率的活细胞中的分子动力学提供了一种强大而灵敏的工具。STED-FCS 提供了在短时间内发生的纳米级分子扩散特性。然而，由于 STED 过程中对荧光的不完全抑制，STED-FCS 点扩散函数 (PSF) 偏离了高斯形状，并对 FCS 获得的自相关曲线的分析提出了挑战。在这里，我们模拟了 STED-FCS 实验中不完全荧光抑制的影响，并提出了一种新的拟合模型，以提高扩散时间和平均分子数测量的准确性。在商用共聚焦/FCS 显微镜上实施带有脉冲激光源的 STED 模块使我们能够应用 STED 背景校正模型来拟合 STED-FCS 测量。实验结果与理论分析一致，分子数和扩散时间随STED功率的增加而相应减少。