Radial analysis of NFRET amplitude distributions. (A) Exemplar NFRET distribution with bins colored by percentile NFRET amplitude. The colors of the pixels in each bin were applied to the NFRET images as displayed in B. The right-hand panel displays how the radius of each pixel from the cell's center of mass was determined (see Materials and methods). The indentation shows that the analysis provides reliable data despite dark areas such as the nucleus or, in some cases, irregular-shaped cells. (B) NFRET images colored by NFRET amplitude percentile for exemplar cells expressing the wild-type–like functioning mGAT1XFP8, efficiently oligomerized but intracellularly retained mGAT1⁵⁶⁵XFP⁵⁶⁶CT, and the poorly oligomerized and predominantly intracellular mGAT1XFP45. The color code for the NFRET amplitude by percentile is at the right. (C) Plot of the mean distance from the center of mass of each percentile group for each construct. Each construct displayed a unique profile. Y error bars, SEM; x bars, size of each percentile group.