(A) HEK293T/17 cells expressing low numbers of TRPV1-GFP are initially imaged for 15 s and then a second 15 s video follows perfusion with a 1 μM capsaicin solution (described in ‘Materials and methods’). All three treatment conditions begin in HBSS (containing Ca2+) with no capsaicin. Average MSD (time lag of 90 msec) for all tracks shows no significant change in mobility from before to after capsaicin. (B) Using the same source data, we characterize mobility with the MSD difference ratio (RΔMSD, described in ‘Materials and methods’ and Figure 6—figure supplement 1). With the switch from no capsaicin to 1 μM capsaicin but maintaining the Ca2+ concentration at 1.8 mM, the RΔMSD drops a significant amount from the initial condition (N = 5, blue data points) as determined by a one-tailed paired t-test (p < 0.01). This significant drop in RΔMSD from the initial video to the second video is not observed under conditions where the second solution contains no capsaicin and no added Ca2+ (N = 6 cells, green data points; n.s.: not significant) nor is it seen when the second solution contains capsaicin (1 μM) but no added Ca2+ (N = 6 cells, red data points).