Supplementary Figure 2: Alternative recording mode and channel calibration for dual-color TOCCSL, TCR surface density distribution measured with KJ25-Fabs and single-molecule FRET events in dual-color TOCCSL measurements.

a, Alternative recording mode involving a dual-color TOCCSL image sequence using alternating red and green excitation (shown is a representative experiment). This imaging modality allows not only for dual-color-based colocalization analysis in the TOCCSL image (images i and ii) but may also provide direct evidence of molecular proximities via FRET upon exciting the FRET donor and imaging the FRET acceptor channel (image iii). The dashed area shows the position of the field stop. b–d, Channel calibration for dual-color TOCCSL experiments. b, Left, the positions of fluorescent multicolor beads detected in the red and green emission channels were overlaid without correction. Right, using this information to calculate the relative shift and stretch of one of the two color channels with respect to the other, the positions of the green color channel were corrected and overlaid with the positions of beads in the red channel. c, Histograms of the positional accuracies (PA) of bead positions in the respective channel are shown. The virtual distance of beads after correction (b, right) is shown to the right and yields an average value of about 20 nm. d, Shown is an example of the distribution of virtual distances for the dual-color TOCCSL experiment with H57-scFv and KJ25-Fab shown in Fig. 2. e, TCR surface densities as measured with KJ25-Fabs. An alternative epitope present on the 5C.C7 TCRβ chain, which is involved in pMHC binding, is probed with the use of KJ25-Fabs. T cells were labeled with AF488-KJ25-Fab and AF647-KJ25-Fab in a 1:1 ratio and placed on a lipid bilayer featuring ICAM-1. The surface densities were calculated for both probes as described in Fig. 1a, yielding an overall TCR surface density of 76 ± 16 molecules per µm². f, Dual-color TOCCSL analysis combined with single-molecule FRET analysis to score for molecular proximity. T cells were simultaneously decorated with AF647-H57-scFv and AF488-KJ25-Fab, and a dual-color colocalization-based TOCCSL experiment was performed. The images were recorded 10 s after the bleach pulse. Positions of diffraction-limited spots were determined for both color channels and are marked with red open circles in the red AF647-H57-scFv channel (top left, red excitation), green open circles in the AF488-KJ25-Fab channel (bottom left, green excitation) and yellow circles in the FRET image (top right, green excitation). Corrected positions of colocalized probes projected into the other color channels are shown as green, red and yellow crosses. For coincidence analysis, the region of interest (ROI) was chosen to ensure the presence of only detection-limited signals (dotted line). Shown is one colocalization (yellow arrow), which is further supported by the presence of a FRET signal (a representative experiment is shown). Scale bars, 4 μm.