Fluorescence methods with single molecule sensitivity have been incredibly improved in the past two decades. These novel approaches have recently provided dynamic insights into the biomolecular mechanisms of molecular motors, ligand binding and protein–nucleic acid interactions. The sensitivity and precision of such *in vitro* fluorescence-based imaging techniques can be further enhanced through the use of nanostructures, such as zero mode waveguides. The increasing time-range and robustness of single molecule techniques, which emerge as a set of crucial tools for elucidating biological processes at utmost detail and accuracy will be demonstrated on our current research on SecYEG driven protein translocation.