Single-molecule fitting and tracking were performed as previously described (9). Briefly, single-molecule puncta of pMHC above a size and intensity threshold were identified using particle detection and tracking algorithm (80) adapted for MATLAB (MathWorks, Natick, MA) as described (81). Single-molecule trajectories were verified using single-step unbinding determined impartially by a Bayesian change point detection algorithm (82). The number and molecular dwell time distributions of the bound pMHC were extracted from the tracks and corrected for photobleaching using data for fluorescent pMHC on a bilayer acquired at the same imaging conditions (illumination power, exposure time, and frame rate).

NFAT translocation was determined by measuring the fluorescence intensity in the cytosol and nucleus. For each cell, the cytosol and nucleus were identified and masked off as different regions using the ISODATA thresholding method in the DIPImage toolbox (83). All images with the masked regions were inspected. The time point at which the nuclear NFAT intensity is first detected to be above the background was set as the time point of initial NFAT translocation. The cumulative binding events is the sum of single-molecule–bound pMHCs that have been detected from cell landing to the point of initial translocation, as explained above, whereas the cumulative dwell time is the sum of the lifetimes of all bound pMHC over the same time period.

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