Supplementary Figure 4: Label-density-variation SMLM of CD3ε labeled with KT3-AF647 and CD3ζ-PS-CFP2.

(a) Representative diffraction-limited microscopy images (left) and dSTORM localization maps (right) of fixed primary murine CD4⁺ T_EFF cells labeled with KT3-AF647 during interaction with non-activating (top) or activating (bottom) supported lipid bilayers (n = 10 and n = 19 biologically independent samples for activating and non-activating conditions, respectively); Scale bars: 3 µm. (b) Normalized ρ versus η plot derived from label density variation dSTORM of fixed primary murine CD4⁺ T_EFF cells under non-activating (blue) and activating (black) conditions using KT3-AF647 (0.02, 0.2, 2, 10 and 20 µg/ml); data were binned based on η with a bin size of 0.1 and represented as means ± SEM; n = 58 for non-activating and n = 20 for activating conditions. Data for individual cells are shown in gray and light blue; red line with pink shaded region indicates reference line and its uncertainty, respectively, for a random distribution derived from simulations based on the experimentally determined blinking statistics of KT3-AF647 (mean ± SEM; n = 50 independent simulations); red arrows indicate data points corresponding to the cells shown in a; Top left, cartoon to illustrate the labeling strategy. (c) Label-density-variation dSTORM using KT3-AF647. Data are identical to b with each data point color-coded according the antibody concentrations used for labeling; Red line with pink shaded region indicates reference line and its uncertainty for a random distribution derived from simulations based on the experimentally determined blinking statistics of KT3-AF647 (mean ± SEM; n = 50 independent simulations). (d) Live primary murine CD4⁺ T_EFF expressing CD3ζ-PS-CFP2 under non-activating conditions. Representative map of all recorded localizations is shown (n = 29 biologically independent samples. Scale bars: 3 µm. (e) Normalized ρ versus η plot derived from label density variation PALM based on the intrinsic expression level variabilities of primary murine CD4⁺ T_EFF cells expressing CD3ζ-PS-CFP2 under non-activating (blue) conditions; data were binned based on η with a bin size of 0.1 and represented as means ± SEM; n = 29. Data for individual cells are shown in light blue; red line with pink shaded region indicates reference line and its uncertainty, respectively, for a random distribution derived from simulations based on the experimentally determined blinking statistics of PS-CFP2 (mean ± SEM; n = 50 independent simulations); red arrow indicates the data point corresponding to the cell shown in d; Top left, cartoon to illustrate the labeling strategy