Fig. 4. (A) Single molecule detection on cells by NSOM. This figure shows a 40 nm optical resolution near-field ‘zoom-in’ on the indicated area (3.2 x 3.2 µm²) in the bright-field image of a fibroblast expressing LFA-1-GFP. GFP excitation is accomplished using 488 nm light (Ar-Kr laser line) linearly polarized along 90°. Fluorescence is collected by a 1.3 numerical aperture oil-immersion objective in combination with standard optical filters. A polarizing beam-splitter cube (Newport, Fountain Valley, CA) is used to split the fluorescence signal into two perpendicular polarized components (compare with Fig. 2). Both signals are then detected by photon-counting avalanche photodiode detectors (APD, SPCM-100, EG&G Electro optics, Quebec). The red/green color-coding of the signals reflects the orientation of the GFP molecules in the plane of the sample. Examples of clustered molecules (arrows) as well as examples showing clear single-molecule detection sensitivity are indicated (circles and squares). The squares show the fast-blinking behavior typical of single molecule GFP fluorescence. The circles present demonstrations of discrete photodissociation phenomena. (B) Estimation of the resolution in the near-field image. This figure shows a line trace through the feature marked with the hexagon in the near-field image. The full width at half maximum (FWHM; arrows) of such traces can be used to obtain an estimate for the maximal resolution (half the FWHM) in the near-field image. On this basis, we estimate the resolution in the near-field image to be 40 nm.

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