Brightness correlation analysis of TRPV1-T468tag/Tet3-Bu-GFP expressing cells and labeled with sTCO-Cy5. 100× objective images of HEK293T cells (DIC, top left) show varied degrees of GFP fluorescence from construct expression (GFP filter image, top middle) and correspondingly have different intensities of membrane labeling by sTCO-Cy5 (Cy5 filter image, top right). ROIs selected for mean GFP fluorescence in five cells (bottom middle) and Cy5 maximal pixel intensity (bottom right) are shown to illustrate our approach of capturing the full cell area to collect GFP fluorescence and selecting membrane regions of Cy5 that are representative of even, homogeneous dye distribution.