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The imaging FCS diffusion law in the presence of multiple diffusive modes
Methods ( IF 4.2 ) Pub Date : 2018-05-01 , DOI: 10.1016/j.ymeth.2017.11.016
Sapthaswaran Veerapathiran , Thorsten Wohland

The cellular plasma membrane is the barrier over which cells exchange materials and communicate with their surroundings, and thus plays the central role in cellular sensing and metabolism. Therefore, the investigation of plasma membrane organization and dynamics is required for understanding of cellular functions. The plasma membrane is a heterogeneous matrix. The presence of structures such as lipid and protein domains and the cytoskeleton meshwork poses a hindrance to the free diffusion of membrane associated biomolecules. However, these domains and the cytoskeleton meshwork barriers are below the optical diffraction limit with potentially short lifetimes and are not easily detected even in super-resolution microscopy. Therefore, dynamic measurements are often used to indirectly prove the existence of domains and barriers by analyzing the mode of diffusion of probe molecules. One of these tools is the Fluorescence Correlation Spectroscopy (FCS) diffusion law. The FCS diffusion law is a plot of diffusion time (τd) versus observation area. For at least three different diffusive modes - free, domain confined, and meshwork hindered hop diffusion - the expected plots have been characterized, typically by its y-intercept (τ0) when fit with a linear model, and have been verified in many cases. However, a description of τ0 has only been given for pure diffusive modes. But in many experimental cases it is not evident that a protein will undergo only one kind of diffusion, and thus the interpretation of the τ0 value is problematic. Here, we therefore address the question about the absolute value of τ0 in the case of complex diffusive modes, i.e. when either one molecule is domain confined and cytoskeleton hindered or when two molecules exhibit different diffusive behavior at the same position in a sample. In addition, we investigate how τ0 changes when the diffusive mode of a probe alters upon disruption of domains or the cytoskeleton by drug treatments. By a combination of experimental studies and simulations, we show that τ0 is not influenced equally by the different diffusive modes as typically found in cellular environments, and that it is the relative change of τ0 rather than its absolute value that provides information on the mode of diffusion.

中文翻译:

多种扩散模式下的成像FCS扩散规律

细胞质膜是细胞交换物质和与周围环境交流的屏障,因此在细胞传感和代谢中起着核心作用。因此,了解细胞功能需要研究质膜组织和动力学。质膜是异质基质。脂质和蛋白质结构域以及细胞骨架网状结构等结构的存在阻碍了膜相关生物分子的自由扩散。然而,这些域和细胞骨架网状屏障低于光学衍射极限,寿命可能很短,即使在超分辨率显微镜下也不容易检测到。所以,动态测量通常用于通过分析探针分子的扩散模式来间接证明域和障碍的存在。这些工具之一是荧光相关光谱 (FCS) 扩散定律。FCS 扩散定律是扩散时间 (τd) 与观察面积的关系图。对于至少三种不同的扩散模式 - 自由、域受限和网状阻碍跳跃扩散 - 预期图已被表征,通常通过其与线性模型拟合时的 y 截距 (τ0),并在许多情况下得到验证。然而,只对纯扩散模式给出了 τ0 的描述。但在许多实验案例中,蛋白质只会经历一种扩散并不明显,因此对 τ0 值的解释是有问题的。这里,因此,我们解决了在复杂扩散模式的情况下 τ0 绝对值的问题,即当任一分子受域限制且细胞骨架受阻时,或者当两个分子在样品中的同一位置表现出不同的扩散行为时。此外,我们研究了当探针的扩散模式因药物治疗破坏结构域或细胞骨架而改变时 τ0 如何变化。通过实验研究和模拟的结合,我们表明 τ0 不受蜂窝环境中常见的不同扩散模式的同等影响,并且是 τ0 的相对变化而不是其绝对值提供了有关模式的信息扩散。当任一分子受结构域限制且细胞骨架受阻时,或者当两个分子在样品中的同一位置表现出不同的扩散行为时。此外,我们研究了当探针的扩散模式因药物治疗破坏结构域或细胞骨架而改变时 τ0 如何变化。通过实验研究和模拟的结合,我们表明 τ0 不受蜂窝环境中常见的不同扩散模式的同等影响,并且是 τ0 的相对变化而不是其绝对值提供了有关模式的信息扩散。当任一分子受结构域限制且细胞骨架受阻时,或者当两个分子在样品中的同一位置表现出不同的扩散行为时。此外,我们研究了当探针的扩散模式因药物治疗破坏结构域或细胞骨架而改变时 τ0 如何变化。通过实验研究和模拟的结合,我们表明 τ0 不受蜂窝环境中常见的不同扩散模式的同等影响,并且是 τ0 的相对变化而不是其绝对值提供了有关模式的信息扩散。我们研究了当探针的扩散模式因药物治疗破坏域或细胞骨架而改变时 τ0 如何变化。通过实验研究和模拟的结合,我们表明 τ0 不受蜂窝环境中常见的不同扩散模式的同等影响,并且是 τ0 的相对变化而不是其绝对值提供了有关模式的信息扩散。我们研究了当探针的扩散模式因药物治疗破坏域或细胞骨架而改变时 τ0 如何变化。通过实验研究和模拟的结合,我们表明 τ0 不受蜂窝环境中常见的不同扩散模式的同等影响,并且是 τ0 的相对变化而不是其绝对值提供了有关模式的信息扩散。
更新日期:2018-05-01
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