Kettmayer Constanza, Gratton Enrico, Estrada Laura C
Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de Física. Buenos Aires, Argentina.
CONICET-Universidad de Buenos Aires, Instituto de Física de Buenos Aires (IFIBA). Buenos Aires, Argentina.
Methods Appl Fluoresc. 2023 Oct 12;12(1). doi: 10.1088/2050-6120/acfd7e.
Fluorescence microscopy can provide valuable information about cell interior dynamics. Particularly, mean squared displacement (MSD) analysis is widely used to characterize proteins and sub-cellular structures' mobility providing the laws of molecular diffusion. The MSD curve is traditionally extracted from individual trajectories recorded by single-particle tracking-based techniques. More recently, image correlation methods like iMSD have been shown capable of providing averaged dynamic information directly from images, without the need for isolation and localization of individual particles. iMSD is a powerful technique that has been successfully applied to many different biological problems, over a wide spatial and temporal scales. The aim of this work is to review and compare these two well-established methodologies and their performance in different situations, to give an insight on how to make the most out of their unique characteristics. We show the analysis of the same datasets by the two methods. Regardless of the experimental differences in the input data for MSD or iMSD analysis, our results show that the two approaches can address equivalent questions for free diffusing systems. We focused on studying a range of diffusion coefficients between D = 0.001msand D = 0.1ms, where we verified that the equivalence is maintained even for the case of isolated particles. This opens new opportunities for studying intracellular dynamics using equipment commonly available in any biophysical laboratory.
荧光显微镜可以提供有关细胞内部动力学的有价值信息。特别是，均方位移（MSD）分析被广泛用于表征蛋白质和亚细胞结构的流动性，从而得出分子扩散规律。传统上，MSD曲线是从基于单粒子跟踪技术记录的单个轨迹中提取的。最近，像iMSD这样的图像相关方法已被证明能够直接从图像中提供平均动态信息，而无需对单个粒子进行分离和定位。iMSD是一种强大的技术，已在广泛的空间和时间尺度上成功应用于许多不同的生物学问题。这项工作的目的是回顾和比较这两种成熟的方法及其在不同情况下的性能，以深入了解如何充分利用它们的独特特性。我们展示了用这两种方法对相同数据集的分析。无论MSD或iMSD分析的输入数据在实验上存在差异，我们的结果表明这两种方法可以解决自由扩散系统的等效问题。我们专注于研究一系列介于D = 0.001m²/s和D = 0.1m²/s之间的扩散系数，在此我们验证了即使对于孤立粒子的情况，等效性也得以保持。这为使用任何生物物理实验室中常见的设备研究细胞内动力学开辟了新机会。
