Supplementary Figure 1: Analysis of H57-scFv binding lifetime, TCR diffusion, anisotropy, TCR diffusion under lactose treatment, target binding of antibody probes and distribution of TCR diffusion constants.

a, The half-life of AF647-H57-scFv bound to surface-exposed TCRs was determined as follows: T cells were labeled under saturating conditions with AF647-H57-scFv and placed onto a lipid bilayer featuring the adhesion molecule ICAM-1. The background-subtracted mean brightness values of individual T cells were determined in TIR mode at various time points (black open circles). Acquired intensities were determined for three time intervals, 0–200 s (n = 11 cells), 800–1,050 s (n = 12 cells) and 1,750–2,000 s (n = 17 cells), and average values were calculated (red open square). The resulting curve was fitted to a single-exponential function and yielded a half-life t½, of 44 ± 9 min at room temperature. b, TCR diffusion constants were determined as follows: TCRs were labeled with AF647-H57-scFv and single-molecule tracking experiments were performed. Mean square displacements were determined and are plotted as a function of time lags. Assuming pure Brownian motion, a linear fit yielded a diffusion coefficient D of 0.037 ± 0.002 μm²/s (n = 21 cells). c, The immobile fraction of TCRs was determined as follows: single-molecule tracking displacements for various time lags between 20 and 140 ms were fitted by a biexponential distribution function (Methods, “TOCCSL analysis”), yielding the fraction of immobile/slowly diffusing TCRs (left; average fraction f = 0.36 ± 0.03), the mean square displacement (msd)–time lag plot of immobile/slowly diffusing TCRs (middle; D = 0.003 μm²/s) and the msd–time lag plot of fast diffusing TCRs (right; D = 0.047 μm²/s). The fraction of fast/mobile TCRs is given by 1 – f = 0.64 ± 0.03 (n = 21 cells). d, Anisotropy measurements: T cells were decorated with the indicated probes at saturating conditions for anisotropy measurements as described in the Methods. For each labeling condition, more than 11 cells were used for the analysis. Anisotropy of all four employed fluorescently labeled pMHCs was measured in bilayers decorated with the respective probe (n = 20 lipid bilayer regions measured per pMHC labeling variant). e, Lactose treatment. T cells were labeled under saturating conditions with AF555-H57-scFv and incubated in imaging buffer, imaging buffer containing 20 mM lactose or imaging buffer containing 20 mM sucrose for 20 min at 4 °C. T cells were then placed onto a lipid bilayer featuring the adhesion molecule ICAM-1. Depending on the carbohydrate pretreatment, FRAP experiments were conducted in the presence of imaging buffer (n = 21 cells), imaging buffer containing 2 mM lactose (n = 16 cells) or imaging buffer containing 2 mM sucrose (n = 16 cells). Mobile fractions for lactose- and sucrose-treated T cells were normalized with regard to the mobile fraction of untreated T cells. f, Target binding of employed probes. T cells were exposed to the probes shown at increasing concentrations and their mean fluorescence intensity (MFI) was determined by flow cytometry. MFIs were normalized to those obtained with the highest probe concentration at which probe saturation had occurred (circles). Data were fitted with an exponential function (solid line). Concentrations, at which half of the TCRs were labeled by the respective probes, yielded 1.83 μg/ml, 3.5 μg/ml and 27.3 μg/ml for H57-scFv, KT3-scFv and KJ25-Fab, respectively (n = 10,000 cells per data point). g, Distribution of diffusion coefficients. Single-molecule tracks from b were analyzed individually, and for every trajectory the diffusion coefficient was determined by mean square displacement analysis (n = 1,260 trajectories). Error bars, s.e.m. (with the exception of a, s.d.).