Halo-PEG coverslips were incubated with 100 nM unlabeled RGD sensor at room temperature for 30 min, and cells were seeded and allowed to spread for at least ~1 hour. After a 9-min incubation with 50 nM SiR-actin (Cytoskeleton Inc., no. CY-SC001), the sample was incubated with Prolong Live Antifade Reagent (Invitrogen, P36975) for 1 hour. The low dilution of SiR-actin allowed for individual molecules to be tracked, and the addition of Prolong reduced photobleaching. For each cell, a 100-ms exposure of the GFP-paxillin channel (for masking) was first acquired, followed by a 60-frame sequence in the far red channel (SiR-actin) with 300-ms exposures taken every 2 s.

For fixed cell control data, cells were allowed to spread on functionalized coverslips and then fixed in 4% paraformaldehyde for 15 min at room temperature. After rinsing, the cells were treated with SiR-actin and Prolong reagent as above.

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