Combined setup and tip-induced quenching imaging. In the combined AFM/fluorescence experiments, the AFM tip is aligned with the laser focus in the center of the microscope objective (a). When the scanning motion moves a fluorescent molecule into the laser focus, fluorescence can be detected (1 in (b) and inset in (a)). As the molecule approaches the AFM tip, the fluorescence is quenched (2). A sharp dip appears in the section through a single molecule fluorescence spot in (b). (c) Fluorescence intensity data of a sample containing single dye molecules with the AFM tip approached to the surface. Diffraction limited spots with black dots close to the center are visible. (d) Smaller scan range of the same sample. (e) The same individual molecules are imaged with retracted tip. The comparison between the scans in (d) and (e) demonstrates that the influence of the AFM tip is well localized and appears as black dots in the diffraction limited spots. The scale bar for (c) is 2 μm and for (d) and (e) 500 nm.