CARs distribute similarly to TCRs on both microvilli and cell cortex, but do not obligately co-localize. (A) Fixed anti-HER2 CAR T cell showing maximum intensity projection of TCR, CAR, CD45 and overlay imaged by lattice light-sheet. Scale bar = 2 μm. Human CD8⁺ T cell expressing anti-HER2 CAR (mut4D5) was labeled with antibodies to MYC (CAR)-Alexa488, TCR (OKT3)-APC, and CD45-Alexa594. (B) Region of cell membrane, as described in A, where MV tips are marked as being high for TCR (red arrow), CAR (green arrow), or both (yellow arrow). Scale bar = 1 μm. (C) 41 tips across 5 regions and 3 cells were scored as in B. (D) Left panel shows zoomed out membrane region in extended view for multiple z slices of a cell labeled as described in A (scale bar = 3 μm). Middle panel shows zoom in on outlined region (scale bar = 1 μm). Right panel shows the same zoom region with the perspective rotated to show the YZ plane (scale bar = 1 μm). (E) Large panels show CD45 surface (gray) overlaid with TCR (left) and CAR (right) surfaces marking areas of high intensity (green). Arrows mark examples of patches on MV tips (white) or cell cortex (yellow). Inset panels show a region of the same cell with the raw TCR and CAR intensity in Fire colorscale. Scale bar = 4 μm. (F) Left: Schematic showing the cell boundary as mapped to radial intensity profile by angle (x-axis) and pixel intensity (y-axis). Right: Pixel intensity (gray line) and binned intensity (black line), defined by taking a moving average of 10 pixels (∼3°), showing one z-slice for TCR, CAR, and CD45. Dashed line with yellow above indicates 3 standard deviations (+3SD) above mean pixel intensity. P indicates binned intensity peaks above +3SD threshold (patches). (G) Number of patches/z-slice for each receptor was defined by the number of excursions of binned intensity above the +3SD threshold. Tukey’s multiple comparisons test was performed on data compiled from multiple slices of 3 cells (n = 17 slices per group). Error bar represents SD.