Reduced mobility of single TRPV1exCellHalo channels after capsaicin treatment. (A) DIC image of HEK293T/17 cells that transiently express TRPV1exCellHalo. (B) TIRF microscopy image of the cell is shown in panel A after labeling TRPV1exCellHalo with Alexa488 HaloTag ligand. The image shown is the first frame of a 2.5-s movie acquired after pre-capsaicin treatment that reveals individual channels diffusing laterally across the cell surface (See Video 1). Overlaid onto the image is the ROI (yellow) to define the area in which ion channels are tracked and individual tracks obtained during the movie. (C) Cumulative density histogram of individual channel track displacements. The total number of tracks is normalized to 1.0 in order to compare displacement distributions from tracks in a movie acquired pre-capsaicin (blue; tracks of panel B and Video 1) to tracks acquired in a second (red) movie 15 s after rapid buffer exchange with a 5 μM capsaicin solution. Dashed, black line guides the eye to the upper-quartile (0.75) value in each distribution. (D) Quantitative comparison between track displacements from pre-capsaicin movies and post-capsaicin movies are made by evaluating the difference in the upper-quartile (0.75) displacement value (see the full set of distributions in Fig. S6). Mock treatment experiments with rapid buffer exchange that contains no capsaicin and day in which the cells are replated onto coverslips for the experiment are noted in table. Statistical significance between pre- and post-capsaicin treatment is determined by paired Student’s t test. (E) Transient expression of TRPV1exCellHalo in isolated mouse DRG culture followed by labeling with Alexa660 HaloTag Ligand.