The NSOM/QD-based system conferred approximately 50-nm resolution scale fluorescence imaging of QD-bound γδ TCR on cell membrane of nonstimulated Vδ2 T cells. (A) Low-magnification NSOM topographic and (B) fluorescence images of 7 nonstimulated blood lymphocytes. Vδ2 TCR⁺ T cells are indicated with a smiley face. (C) The high-magnification NSOM fluorescence and topographic (inset) images of the cell marked by a smiley face as shown in panel A. (D) The fluorescence intensity profile of the cross section between the 2 fluorescent objects marked by 2 arrows in panel C. The short arrow shows an approximately 41-nm fluorescence dot representing a 1-QD–bound TCR dot, and the long arrow shows an approximately 80-nm fluorescence dot possibly corresponding to 2-QD-bound TCR cluster. (E) The NSOM fluorescence images enlarged from an area in panel C (inset: the corresponding topographic image). The higher-magnification imaging shows immunofluorescence images of individual 1× approximately 50-nm TCR dots (short arrows), 2× approximately 50-nm TCR cluster (long arrow), and more than 2× approximately 50-nm TCR clusters (dotted circles). (F) The fluorescent intensity profile of the cross section of the objects marked by a dashed line in panel E. Scan size: (C) 11 × 11 μm²; (E) approximately 1 × 1 μm². Resolution: (C) 500 × 500 pixel²; (E) 400 × 400 pixel². Integration time: (C) 15 ms; (E) 10 ms. (G) The left histogram showing the frequency or distribution for the immunofluorescence FWHM of individual Ab-QD–bound TCR (n = 326) on cell membrane of cells; the right histogram showing fluorescence intensity distribution of Ab-QD–bound TCR on the same cells. Mean FWHM diameters of the QD-Ab-bound TCR were 53.7 plus or minus 18.9 nm (mean ± SD), with approximately 50-nm dots being predominant (left histogram); the scale 20 was the dominant fluorescent intensity count of the individual QD-Ab-bound TCR on the cell membrane of cells (right histogram). Similar to what is seen in Figure 1E, individual QD-Ab–bound TCR dots exhibited a good relationship between approximately 50-nm dots and 20 intensity counts (basic-scale fluorescence) on the cell membrane (< 10 intensity scales were background). The excitation condition was the same as that described in Figure 1E. Scan size: 500 nm × 500 nm. Resolution: 300 pixel × 300 pixel. Integration time: 10 ms. The imaging was derived from Ab-conjugated QD655 as the second Ab. Similar results were seen when using QD655-streptavidin.