Figure 2

Resolution and mechanism of tip-induced quenching imaging. Quenching of single Atto655 molecules by the AFM tip is highly localized. For a sharp probe, the full width at half maximum (FWHM) of the quenched spot can be less than 5 nm (a). For (b), (c), (d) the FWHMs are 6.8 nm, 7.5 nm, and 5.9 nm, respectively. For blunt tips, the shape of the end of the tip can be clearly seen as a triangle (e). In (f) the fluorescence lifetime distribution, the calculated fluorescence intensity and the measured fluorescence intensity of a quenching spot with a FWHM of 16 nm is shown. The distribution of fluorescence intensities and lifetimes gives insight into the physical mechanisms that lead to quenching. The effect of the AFM tip on a fluorophore depends on the orientation and distance between them (g). For very small separations, parallel orientation leads to out-of-phase oscillation of the molecular dipole and its mirror image (g, middle). In the case of normal orientation (g, right), the dipole oscillates in phase. The scale bars in (a)-(f) are 50 nm.