Traction Force Microscopy Using an Epifluorescence Microscope: Experimental Considerations and Caveats

Forces exerted by cells due to their internal contractility play fundamental roles in a host of processes, including adhesion, migration, survival and differentiation. Traction force microscopy (TFM) enables the determination of forces exerted by cells or cell collectives on their environment, which is typically taken to be an extra-cellular matrix (ECM)-coated substrate. Sample preparation for TFM involves the plating of cells onto an environment embedded with fiducial markers. The imaging of these fiducial markers in the presence and absence of the cells then enables calculation of the displacement of localized regions of the environment, and, consequently, the spatial distribution of forces exerted by the cells on their environment. Here, we consider the most widely used implementation of TFM (two-dimensional or 2D TFM) which enables the determination of in-plane forces exerted by cells plated on top of an elastic soft substrate. We present streamlined methods for preparing TFM substrates, with special consideration towards experimental steps involved in implementing it using an epifluorescence microscope. We highlight considerations involved in substrate choice between polyacrylamide (PAA) gels and soft silicones, fiducial marker (microbead) choice and distribution as well as microbead and ECM coupling to the substrate. We also point out caveats related to sub-optimal choices in the methodology which can affect the resultant traction force distribution, as well as further derived quantities such as inter-cellular forces in cell pairs computed using the traction force imbalance method (TFIM).