Traction force microscopy (TFM) derives maps of cell-generated forces, typically in the nanonewton range, transmitted to the extracellular environment upon actuation of complex biological processes. In traditional approaches, force rendering requires a terminal, time-consuming step of cell deadhesion to obtain a reference image. A conceptually opposite approach is provided by reference-free methods, opening to the on-the-fly generation of force maps from an ongoing experiment. This requires an image processing algorithm keeping the pace of the biological phenomena under investigation. Here, we introduce an integrated software pipeline rendering force maps from single reference-free TFM images seconds to minutes after their acquisition. The algorithm tackles image processing, reference image estimation, and finite element analysis as a single problem, yielding a robust and fully automatic solution. The method's capabilities are demonstrated in two applications. First, the mechanical annihilation of cancer cells is monitored as a function of rising environmental temperature, setting a population threshold at 45 °C. Second, the fast temporal correlation of forces produced across individual cells is used to map physically connected adhesion points, yielding typical lengths that vary as a function of the cell cycle phase.
Keywords: Real time analysis; cTFM; focal adhesion; reference free; stress fibers; traction force microscopy.