NSOM/QD-based imaging showed that Vγ2Vδ2 TCR arrayed to form high-density TCR nanoclusters, nanodomains, and microdomains during the in vivo clonal expansion of Vγ2Vδ2 T cells after HMBPP/IL-2 treatment. (A) Representative NSOM images of TCR nanoclusters, nanodomains, and microdomains on the membrane of clonally expanded Vδ2 T cells on day 4. (Ai) NSOM topographic images of 3 closely adjacent cells as marked by a smiley face in the low-magnification images showing 6 closely adjacent cells in panel Aiii. (Aii) The NSOM fluorescence image displaying the polarized γδ TCR nanoclusters, nanodomains, and microdomains on the 2 corresponding Vδ2 T cells as shown in panel Ai. (Aiv) The enlarged NSOM fluorescence image of TCR nanoclusters, nanodomains, and microdomains on the Vδ2 T cell shown in the upper left of panel Aii. (B) Representative NSOM topographic (left) and fluorescence (middle) images indicating the dominance of nonengaging fluorescence TCR dots on the membrane of unstimulated Vγ2 T cells on day 0. The fluorescent intensity profile graph (right) is extracted from a random cross section in the fluorescence image (middle) showing that predominant fluorescence TCR dots here displayed FWHM of approximately 50 nm. (C) Representative NSOM images of TCR nanoclusters, nanodomains, and microdomains on the membrane of clonally expanded Vγ2 T cells on day 4. (Cii) Enlarged from the boxed area in the low-magnification NSOM image panel Ci. The fluorescence intensity profile (Civ) is extracted from the cross-sectional part (dashed arrow in Ciii), which is enlarged from the boxed area in panel Cii. (Cv) The NSOM fluorescence image of another activated/expanded Vγ2 T cell collected on day 4. (D) Boxplot (left) and histogram (right) showing the FWHM of approximately 50-nm TCR dots and TCR nanoclusters/nanodamains/microdomains before and day 4 after HMBPP/IL-2 treatment. (**P < .001 vs the values of the unstimulated cells). In the left panel, the center line is the median, the dot is the mean, the boxes are interquartile ranges, and the whiskers are the value ranges. The histogram on the right shows the percentages of different sizes of TCR clusters in total γδ TCR that were counted and measured. The data are the mean frequencies calculated from 5 Vδ2⁺ and Vγ2⁺ T cells. Scan size: (Aiii, Ci) 25 × 25 μm²; (Ai,ii, Cii) 15 × 15 μm²; (Cv) 11.5 × 11.5 μm²; (Aiv, B) 9 × 9 μm²; (Ciii) 4.5 × 4.5 μm². Resolution: (Aiii, Ci) 300 × 300 pixel²; (Ai, Aii, B) 400 × 400 pixel²; (Aiv, Cii,iii, and Cv) 500 × 500 pixel². Integration time (ms): (Aii-iv) 40; (B) 15; (Ci-iii) 10; (Cv) 20.