The mechanobiology of receptor-ligand interactions and force-induced enzymatic turnover can be revealed by simultaneous measurements of force response and fluorescence. Investigations at physiologically relevant high labeled substrate concentrations require total internal reflection fluorescence microscopy or zero mode waveguides (ZMWs), which are difficult to combine with atomic force microscopy (AFM). A fully automatized workflow is established to manipulate single molecules inside ZMWs autonomously with noninvasive cantilever tip localization. A protein model system comprising a receptor-ligand pair of streptavidin blocked with a biotin-tagged ligand is introduced. The ligand is pulled out of streptavidin by an AFM cantilever leaving the receptor vacant for reoccupation by freely diffusing fluorescently labeled biotin, which can be detected in single-molecule fluorescence concurrently to study rebinding rates. This work illustrates the potential of the seamless fusion of these two powerful single-molecule techniques.
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