T-cells readily detect the presence of even a single antigenic peptide/MHC complex (pMHC) among thousands of endogenous pMHCs via T-cell receptors (TCRs) on the surface of antigen-presenting cells. The mechanisms underlying this phenomenal sensitivity have remained elusive. Recent studies suggest that the topography of the immunological synapse formed between the T cell and the antigen-presenting cell is of pivotal importance for these processes. We hence were first interested, how the TCR is distributed within the immunological synapse. For this, we used single molecule localization microscopy in combination with supercritical angle detection to localize single TCR molecules at an isotropic precision below 15nm [1]. Second, researchers speculated that mechanical forces could be instrumental for the high specificity and sensitivity of the T cell response. Force magnitude, spread, and temporal behavior, however, are still poorly defined. We developed a calibrated FRET-based sensor equipped either with a TCR-reactive single chain antibody fragment or peptide-loaded MHC. The sensor was tethered to planar glass-supported lipid bilayers and informed most directly on the magnitude and kinetics of TCR-imposed forces at the single molecule level. From the single molecule FRET signals we quantified the magnitude of tensile forces exerted by T cells via single TCR molecules [2, 3]. In addition, the data allowed us to draw conclusions on the directionality of the observed forces, as well as on the pulling speed. References 1 Velas, L. et al. Three-Dimensional Single Molecule Localization Microscopy Reveals the Topography of the Immunological Synapse at Isotropic Precision below 15 nm. Nano Lett 21, 9247-9255 (2021). 2 Göhring, J. et al. Temporal analysis of T-cell receptor-imposed forces via quantitative single molecule FRET measurements. Nature Communications 12, 2502 (2021). 3 Schrangl, L. et al. CD4+T-cells create a stable mechanical environment for force-sensitive TCR:pMHC interactions. Nature Communications 16, 7577 (2025).