We show that a concentration of light at a metal tip allows near-field optical imaging of single fluorescent dye molecules at very high resolution, despite strong quenching effects. Details as small as 10 nm were observed in the fluorescence patterns of single Cy-3 dyes bound to the termini of DNA. Data evaluation by model fitting determines the positions of the dyes to an accuracy even better than 1 nm and also yields their 3D orientation. The metal tip simultaneously provides high-resolution topographic imaging complementing the optical signal for a detailed surface examination.

We show improvement of the optical and topographical resolution of scanning near-field optical microscopy by introducing a “tip-on-aperture” probe, a metallic tip formed on the aperture of a conventional fiber probe. The tip concentrates the light passing through the aperture. Thus the advantages of aperture and apertureless scanning near-field optical microscopy are combined. Tips are grown by electron beam deposition and then covered with metal. Fluorescent beads are imaged with a resolution down to 25 nm (full width at half maximum) in the optical signal. The near-field appears strongly localized within 5 nm in z direction, thus promising even higher resolution with sharper tips.

Back-illuminated full body glass tips coated with a thin metal layer can be used as local probes for apertureless scanning near-field optical microscopy (SNOM). In order to achieve high spatial resolution, high electric field intensities and low background illumination, the thickness of the metal coating, angular illumination direction, and polarization have to be optimized. Optimal conditions have been calculated and experimentally verified for 10–15 nm thick aluminium and 15–25 nm thick silver layers. Upon imaging single dye molecules, characteristic single and double-peak patterns with peak widths down to 15 nm could be measured, exhibiting an optical resolution which exceeds the classical diffraction limit of Abbé significantly.

SummaryIn fluorescence microscopy and spectroscopy, energy transfer processes between single fluorophores and fluorophore quencher pairs play an important role in the investigation of molecular distances or orientations. At distances larger than about 3 nm these effects originate predominantly from dipolar coupling. As these experiments are commonly performed in homogenous media, effects at the interface boundaries can be neglected. Nevertheless, the combination of such assays with single-molecule manipulation techniques such as atomic force microscopy (AFM) requires a detailed understanding of the influence of interfaces on dipolar coupling effects. In the presented work we used a combined total internal reflection fluorescence microscopy (TIRFM)–AFM setup to elucidate this issue. We measured the fluorescence emission emanating from single quantum dots as a function of distance from the apex of a gold-coated cantilever tip. As well as fluorescence quenching at close proximity to the tip, we found a nonlinear and nonmonotonic distance dependence of the fluorescence emission. To confirm and interpret our findings we performed calculations on the basis of a simplified multiple multipole (MMP) approach, which successfully supports our experimental data. Moreover, we revealed and quantified the influence of interfering processes such as field enhancement confined at interface boundaries, mirror dipoles and (resonant) dipolar coupling.

Correlated topographic and spectroscopic imaging beyond diffraction limit by atomic force microscopy metallic tip-enhanced near-field fluorescence lifetime microscopy Rev. Sci. Instrum. 74, 3347 (2003); 10.1063/1.1581359Influence of protective layers on the blinking of fluorescent single molecules observed by confocal microscopy and scanning near field optical microscopy