High-density TCR nanoclusters, nanodomains, and microdomains were also developed during the in vivo clonal expansion of Vγ2Vδ2 T cells after the Picostim/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. On the left are topographic images; on the right are fluorescent images. The images in panel Ai-Aiv are sequential magnification images from the boxed area in the image. The mean clusters on the cells at this time point are 132.7 plus or minus 37.1 nm, a nanoscale imaging that cannot be reached by confocal microscopy because of the limited optical resolution (∼300 nm). Scanning sizes: (Ai) 25 × 25 μm²; 300 × 300 pixel²; (Aii) 15 × 15 μm²; 500 × 500 pixel²; (Aiv) 1.5 × 1.5 μm²; 300 × 300 pixel². (Av) Fluorescent intensity profile of the cross section of the objects (the (dashed line in panel Aiv). Shown are 2 TCR clusters with diameters (FWHM) of 56 nm and 114 nm, respectively. Integration time in panels Ai to Aiv: 30 ms. (B) Representative NSOM images of TCR nanoclusters, nanodomains, and microdomains on the membrane of clonally expanded Vδ2 T cells on day 7 after Picostim/IL-2 treatment. Shown are the NSOM topographic (left) and fluorescence (middle) images (16 × 16 μm²; 500 × 500 pixel²; integration time: 30 ms) of a Vγ2Vδ2 T cell. The TCR clusters on the cells at this time point are 315.6 plus or minus 102.4 nm. The fluorescent intensity profile (right) of the cross section of the objects is marked by a line in the fluorescence image (middle). Shown are 2 TCR clusters with diameters of 234 nm and 700 nm (FWHM), respectively. (C) Boxplot (left) and histogram (right) graphs showing the FWHM of approximately 50-nm TCR dots and TCR nanoclusters/nanodamains/microdomains before and days 4 and 7 after Picostim/IL-2 treatment. Box and whisker plot is as described in Figure 5D. (**P < .001 vs the values of the unstimulated cells.) 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⁺ cells.