Quantification of OT-I–GFP T cell surface TCR fluorescence in vivo. (a) TCR expression on the surface of OT-I–GFP Rag2^−/− T cells versus WT T cells with endogenously rearranged Vα2⁺ or Vβ5⁺ TCRs. Data are representative of more than three experiments. (b–d) A localized active contour algorithm was used to identify the cell edges of OT-I–GFP Rag2^−/− cells in LNs from two-photon images. GFP images were masked in the cell surface regions. n = 27 cells from three experiments. (b) Representative cell surface identified by the local contour segmentation. Z-series images of an OT-I–GFP Rag2^−/− cell (top row) with a mask marking the surface-exposed pixels of the cell (green in bottom row). The interplane distance is 1 µm. Bar, 4 µm. (c) Surface volume rendering of an OT-I–GFP cell segmented using the active contour algorithm. (d) Quantification of green fluorescence of OT-I–GFP cells masked on the cell surface voxels.