Guidance for 3D traction force microscopy today and in the next decade.

The field of mechanobiology studies how mechanical forces influence cell behavior, relying on tools like traction force microscopy (TFM) to quantify cell forces exerted on the extracellular matrix. While well established for two-dimensional in vitro systems, its three-dimensional form, 3DTFM, remains underutilized despite notable technical advancements. Here, we outline common skepticism about 3DTFM, detailing current experimental and computational strategies to address its limitations. We describe how to integrate 3DTFM with biological readouts, focusing on its application in long-term experiments. We discuss metrics for data interpretation and how pairing these with optimal traction recovery methods can address specific biological questions. Finally, we outline future directions by proposing combinations with emerging technologies to address challenges like extracellular matrix heterogeneity and intracellular stress analysis within three-dimensional cell clusters. By addressing these critical gaps, this Perspective aims to advance 3DTFM's utility, promote its broader adoption and guide future developments in mechanobiology.