A FRET-based TSMod inserted into the C. elegans β-spectrin gene (unc-70) was used to assess forces in live embryos. (A) Schematic for how UNC-70(TSMod) detects tension. FRET occurs when the donor fluorophore (mTFP) transfers energy to a nearby acceptor fluorophore (Venus) within the same peptide. When UNC-70(TSMod) experiences mechanical tension, a flexible linker separating mTFP and Venus is lengthened, leading to reduced FRET efficiency. (B–D) Representative images of wild-type and (F-H) pha-1(tm3671) strains that express UNC-70(TSMod). (J–L) Representative images of wild-type embryos expressing the no force control UNC-70(N-TSMod). Panels B, F, J depict 1.5-fold embryos after direct excitation of the Venus acceptor fluorophore. Panels C, D, G, H, K, L show FRET measurements where purple pixels indicate regions of highest tension (low FRET). Small white-framed boxes in panels B, C, F, G, J, K indicate the sensory depression region (SDR), which is enlarged in panels D, H, L. Red dashed lines in panels B, C, E, F, J, K outline the embryos. Scale bar in B = 30 µm. (E, I, M) FRET indices for the SDR and the region outside the sensory depression (Out-SDR). Individual embryos are represented by red circles, which are connected by lines to indicate values acquired from the same embryo. p-values depicted were calculated using a T-test (also see Supplementary file 2). Numbers at the bottom indicate the number of embryos that were analyzed for each condition. Each point is an average of ∼3–5 frames from a z-stack encompassing the embryo (see ‘Materials and methods’ for details).