Combined aperture NSOM and fluorescent QD nanotechnology conferred approximately 50-nm resolution scale fluorescence imaging of individual QD655 on glass substrate. (A) Electron micrograph of fluorescent quantum dots (QD655). These disperse semiconductor nanocrystals have a diameter of approximately 10 nm under EM. The EM image was derived from a JEOL JEM-1220 transmission electron microscope using a standard EM protocol. (B) NSOM topographic image of the surface of clean glass substrates (no detectable fluorescence; data not shown). (C) NSOM topographic (left) and corresponding fluorescence (right) images of individual Ab-conjugated QD655 molecules. Scan size: 500 nm × 500 nm. Resolution: 300 pixel × 300 pixel. Integration time: 10 ms. (D) The height (left) and fluorescence intensity (right) profiles of the cross sections corresponding to the white lines on images of panel C. (C,D) The diameters of individual Ab-conjugated QD655 are predominantly approximately 50 nm both in topography and fluorescence intensity, which is consistent with that of individual quantum dots bound to TCR on cell membrane surface as shown in Figure 2. Similar results were seen for streptavidin-conjugated fluorescent QD (data not shown). (E) The left histogram shows the frequency or distribution for the immunofluorescence FWHM of individual Ab-conjugated QD655 molecules (n = 608); the right histogram shows fluorescence intensity distribution of Ab-conjugated QD655 on the same glass substrate. Mean diameters of the individual conjugated QD655 were 51.63 plus or minus 13.46 nm (mean ± SD), with approximately 50-nm dots being predominant (left histogram); the scale 20 was the dominant fluorescent intensity unit of the individual conjugated QD655 on the glass substrate (right histogram). The scale 20 was the weakest fluorescence intensity counts, even on the glass substrate prepared from further dilutions of QDs (≤ 10 intensity scales are background).