Revisiting single-cell migration parameters from 2D video microscopic measurements

Video microscopy-based single-cell migration analysis allows for precise measurement of cell migration by extracting a range of migration parameters from tracking data during analysis. This study aimed to enhance our understanding of the behavior and sensitivity of these parameters themselves. Random walk simulations and analysis of experimental data were utilized to analyze the sensitivity of migration parameters to tracking methods (manual and semi-automatic), and to assess the repeatability of these parameters. Additionally, the influence of cell line-specific characteristics on migration outcomes was examined. Our results demonstrate that displacement-based parameters - mean squared displacement (MSD), displacement (D), and maximal displacement (Max D) - are robust in detecting migration effects but are profoundly influenced by cell line-specific properties, particularly with semi-automatic tracking. These metrics are especially valuable when analyzing data from different, non-identical tracking methods. In contrast, path-based parameters - such as total traveled distance (TTD), velocity (V), and average velocity (AV) - are more sensitive to subtle treatment effects, but are prone to noise from tracking methods and inherently insensitive to treatment-induced changes in directionality. Our findings emphasize the importance of combining path-based and displacement-based metrics in video microscopy analyses to achieve a comprehensive picture of cell migration dynamics.