A new approach for high-content traction force microscopy to characterize large cell ensembles

Abstract

1.1Accurate measurements of cellular forces are important for understanding a wide range of biological processes where traction plays a major role. The characterization of mechanical properties is needed to unravel complex phenomena like migration, morphogenesis, mechanotransduction, or shape regulation, but accurate data on large numbers of single cells remain scarce and challenging. The capacity to measure forces in populations of cells and to identify subsets within heterogeneous ensembles would enable to reveal and manipulate their intrinsic complexity. Traction Force Microscopy (TFM) is a technology that can quantify the contractile forces exerted by cells via measuring the displacement of fluorescent beads embedded on the surface of a soft substrate with precisely defined mechanical properties. However, conventional numerical approaches for measuring cellular forces using TFM are labor-intensive and can yield significant artifacts, making them ill-suited for high-throughput analysis. In this work, we propose using the Demons algorithm instead, leading to significant improvements in both computational efficiency and accuracy. Based computer simulations, we show that in some situations this methodology outperforms conventional approaches in terms of speed, it is less sensitive to the blur induced by out-of-focus images and improves the accuracy of force field reconstructions. Additionally, we conducted experiments using cell lines and gels of distinct stiffness to demonstrate that the Demons algorithm is capable of real-time analysis and is effective at clustering cells according to their mechanotype. 1.2 Statement of significanceLarge cellular ensembles encompass a wide variety of traits that require detailed but high-throughput technologies for characterisation. Granular descriptions of cell populations enable to describe their heterogeneity are essential to understand biological phenomena in the search of effective treatments. Most advances in the field of Traction Force Microscopy (TFM) are oriented towards high resolution force measurements at sub-micron adhesion scale that reveal the intricacies of molecular interactions. Here, we propose a strategy to exploit TFM for characterizing the mechanotypes of large cell ensembles. We benchmark and show the capacities of using the Demons algorithm to measure bead displacement fields, the improvement in force reconstructions obtained, and experimentally demonstrate that single cell analysis of cell dynamics can be used to cluster and describe different cell types.