Progress in nanosciences and life sciences is closely related to developments of high resolution imaging techniques. We introduce a technique which produces correlated topography and fluorescence lifetime images with nanometer resolution. Spot sizes below 5 nm are achieved by quenching of the fluorescence with silicon probes of an atomic force microscope which is combined and synchronized with a confocal fluorescence lifetime microscope. Moreover, we demonstrate the ability to locate and resolve the position of two fluorescent molecules separated by 20.7 nm on a DNA origami triangle with 120 nm side length by correlating topography and fluorescence data. With this method, we anticipate applications in nano-and life sciences, such as the determination of the structure of macromolecular assemblies on surfaces, molecular interactions, as well as the structure and function of nanomaterials.

Progress in nanosciences and life sciences is closely related to developments of high resolution imaging techniques. During the last decades the invention and improvement of scanning probe microscopy techniques like atomic force microscopy (AFM) [1] have opened new views onto nanoscale materials. On the other side, optical microscopy has been pushed beyond the diffraction limit and the observation of single molecules has evolved into a standard technology [2]. We have performed experiments with a combined AFM/fluorescence lifetime setup to evaluate the feasibility of using such a combination for contact mode AFM topographic and super-resolution fluorescence imaging. We show distinct modulation of the fluorescence intensity and lifetime due to AFM probes of different materials in contact mode. Our experimental setup combines an MFP 3D-Bio AFM from Asylum Research, California, with a Microtime200 confocal fluorescence lifetime imaging system from PicoQuant, Germany. The AFM controls the motion of a piezo scanning stage that holds the sample. The scanning motion is synchronized with the data collection of the confocal microscope such that the data from the two instruments can be spatially correlated. The synchronization as well as the alignment of the confocal volume around the AFM probe has been described in detail in [3]. We have used AFM probes of three different materials for combined scans in contact mode. For all tested materials, namely silicon, silicon nitride, and quartz tips, we see a distinct dark spot in the emission from single Atto655 molecules on a glass surface (Fig 1). For silicon probes, we found spot sizes as small as 5nm (FWHM).As samples that provide topographic information as well as fluorescence emission, we immobilized DNA origami triangles modified with Atto655 on mica. Base pairing with the main strand of the DNA ensured that two fluorescent molecules are in a well defined position on the origami triangle. This allows us to compare the topographic image of the DNA triangle with the quenched spot in the fluorescence image, which indicates the position of the Atto655 molecules on the structure. The experimental data (Fig. 2) shows good agreement between the expected location of the fluorophore and the quenching spot in the fluorescence image.