Supplementary Figure 1: T cell activation model systems, quantification of T cell activation by ratiometric Ca²⁺ imaging and titration steps in label-density-variation dSTORM with H57-AF647.

(a) Schematic of model system: primary murine CD4⁺ T_EFF cells on supported lipid bilayers displaying ICAM-1 (green) for non-activating conditions (top), or ICAM-1, B7-1 (red), and cognate pMHCII (blue) for activating conditions (bottom). (b) Ratiometric Ca²⁺ imaging via Fura-2 to assess activation state of primary murine CD4⁺ T_EFF cells. T cell activation and concomitant increase in cytosolic Ca²⁺ under activating conditions (black, 2,128 cells from 7 independent experiments) or non-activating conditions (see a): unlabeled cells (blue, 2,362 cells from 7 independent experiments), or cells labeled with H57-AF647 (magenta, 763 cells from 2 independent experiments), H57-scFv-AS635P (green, 293 cells from 3 independent experiments), or KT3-scFv-AS635P (orange, 1205 cells from 4 independent experiments). Data shown as medians ± SE of the median. (c) Label-density-variation dSTORM using H57-AF647. Data represent one titration experiment shown in Fig. 2b 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 H57-AF647 (mean ± SEM; n = 50 independent simulations)