Abstract
Recent improvements in methods of single-particle fluorescence tracking have permitted detailed studies of molecular motion on the nanometer scale. In a quest to introduce these tools to the burgeoning field of DNA nanotechnology, we have exploited fluorescence imaging with one-nanometer accuracy (FIONA) and single-molecule high-resolution colocalization (SHREC) to monitor the diffusive behavior of synthetic molecular walkers, dubbed "spiders," at the single-molecule level. Here we discuss the imaging methods used, results from tracking individual spiders on pseudo-one-dimensional surfaces, and some of the unique experimental challenges presented by the low velocities (approximately 3 nm/min) of these nanowalkers. These experiments demonstrate the promise of fluorescent particle tracking as a tool for the detailed characterization of synthetic molecular nanosystems at the single-molecule level.