Sub-diffraction-limited imaging of fluorescent monomers on sliding microtubules in vitro by nanoscale localization sampling (NLS) is reported. NLS is based on periodic nanohole antenna arrays that create locally amplified electromagnetic hot spots through surface plasmon localization. The localized near-field hot spot temporally samples microtubular movement for enhanced spatial resolution. A fourfold improvement in spatial resolution compared to conventional wide-field microscopy is demonstrated. The resolution enhancement is achieved by imaging rhodamine-labeled microtubules that are sampled by the hot spots to provide sub-diffraction-limited images at 76 nm resolution in the direction of movement and 135 nm orthogonally. The intensity distribution produced by the NLS is measured to be broader than that of conventional imaging, which is consistent with the improvement of imaging resolution. Correlation studies between neighboring nanoantennas are also performed. This confirms the possibility of measuring microtubular transport dynamics. NLS can be useful for moving objects that have a high labeling density or for performing fluctuation spectroscopy in small volumes, and may allow “super-resolution on demand” by customizing nanoantenna structures for specific resolution needs.