Fluorescence correlation spectroscopy (FCS) is a cornerstone technique in optical microscopy to measure, for example, the concentration and diffusivity of fluorescent emitters and biomolecules in solution. The application of FCS to complex biological systems, however, is fraught with inherent intricacies that impair the interpretation of correlation patterns. Critical among these intricacies are temporal variations beyond diffusion in the quantity, intensity, and spatial distribution of fluorescent emitters. These variations introduce distortions into correlated intensity data, thus compromising the accuracy and reproducibility of the analysis. This issue is accentuated in imaging-based approaches such as pair correlation function (pCF) analysis due to their broader regions of interest compared with point-detector-based approaches. Despite ongoing developments in FCS, attention to systems characterized by a spatiotemporal-dependent probability distribution function (ST-PDF) has been lacking. To address this knowledge gap, we developed a new analytical framework for ST-PDF systems that introduces a dual-timescale model function within the conventional pCF analysis. Our approach selectively differentiates the signals associated with rapid processes, such as particle diffusion, from signals stemming from spatiotemporal variations in the distribution of fluorescent emitters occurring at extended delay timescales. To corroborate our approach, we conducted proof-of-concept experiments on an ST-PDF system, wherein the, initially, uniform distribution of fluorescent microspheres within a microfluidic channel changes into a localized accumulation of microspheres over time. Our framework is offering a comprehensive solution for investigating various phenomena such as biomolecular binding, sedimentation, and particle accumulation.

中文翻译：

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解决配对相关函数分析中的时空信号变化


荧光相关光谱 (FCS) 是光学显微镜中的一项基础技术，用于测量溶液中荧光发射体和生物分子的浓度和扩散率等。然而，FCS 在复杂生物系统中的应用充满了固有的复杂性，损害了相关模式的解释。这些错综复杂的问题中最重要的是荧光发射体的数量、强度和空间分布超出扩散范围的时间变化。这些变化会导致相关强度数据失真，从而影响分析的准确性和可重复性。这个问题在基于成像的方法（例如对相关函数（pCF）分析）中尤为突出，因为与基于点检测器的方法相比，它们的感兴趣区域更广泛。尽管 FCS 不断发展，但人们仍然缺乏对以时空相关概率分布函数 (ST-PDF) 为特征的系统的关注。为了解决这一知识差距，我们开发了一种新的 ST-PDF 系统分析框架，该框架在传统 pCF 分析中引入了双时间尺度模型函数。我们的方法选择性地区分与快速过程（例如粒子扩散）相关的信号和源自延长延迟时间尺度下荧光发射器分布的时空变化的信号。为了证实我们的方法，我们在 ST-PDF 系统上进行了概念验证实验，其中最初荧光微球在微流体通道内的均匀分布随着时间的推移变成了微球的局部积累。 我们的框架为研究生物分子结合、沉降和颗粒积累等各种现象提供了全面的解决方案。