Optical Characteristics of Atomic Force Microscopy Tips for Single-Molecule Fluorescence Applications

Knowledge of the optical properties of atomic force microscopy (AFM) tips is relevant for the combination of optical and force spectroscopy. The luminescence properties of five commercial AFM tips were characterized using a combination of multiparameter fluorescence detection (MFD) and scanning confocal techniques. These include three Si3N4 tips, one silicon tip, and one high‐density carbon (HDC) tip grown on top of a silicon tip. Time‐decay histograms of the signal were analyzed to determine the strength of scatter, constant background, and fluorescence in the observed signal. Intensity and anisotropy images with optical resolution down to the diffraction limit were generated. The optical signal recorded from the apex of the Si3N4 tips ranged from 0.7 to 1.9 times the count rates from single Rhodamine 110 molecules under similar illumination conditions. The signal is predominantly composed of scatter and background (>85 %), plus a small fluorescence component with lifetimes between 1 and 3 ns. The intensity of the recorded signal fell with increasing distance from the apex, and by 300 nm the signals fell below single‐molecule levels for all Si3N4 cantilevers. Silicon cantilevers demonstrated very low count rates relative to single‐molecule measurements under all conditions, and virtually no fluorescence. The high‐density carbon tips also demonstrated low count rates, but the signal contained a short lifetime fluorescence component (0.7 ns). The intensity of the signals from each of the tips was geometry dependent, demonstrating the highest intensities at the edges and corners. Likewise, the anisotropy of all tip signals was observed to be geometry dependent, with the dependence varying on a case‐by‐case basis. The implications for using confocal illumination instead of total internal reflection are discussed. Testing tips: Several commercial atomic force microscopy (AFM) cantilevers are shown to have light scattering and luminescence characteristics suitable for combined single‐molecule AFM/fluorescence experiments. For most of the cantilevers tested, the signal is comparable to the scattering signal from the surrounding buffer when the focus of the optical setup (see figure) is more than 300 nm away from the tip. The cantilevers were investigated with a diffraction‐limited scanning confocal fluorescence microscopy setup.