DNA nanotechnology has developed powerful techniques for the construction of precisely defined molecular structures and machines, and nanoscale imaging methods have always been crucial for their experimental characterization. While initially atomic force microscopy (AFM) was the most widely employed imaging method for DNA-based molecular structures, in recent years a variety of other techniques were adopted by researchers in the field, namely electron microscopy (EM), super-resolution fluorescence microscopy, and high-speed AFM. EM is now typically applied for the characterization of compact nanoobjects and three-dimensional (3D) origami structures, as it offers better resolution than AFM and can be used for 3D reconstruction from single-particle analysis. While the small size of DNA nanostructures had previously precluded the application of fluorescence microscopic methods, the development of super-resolution microscopy now facilities the application of fast and powerful optical methods also in DNA nanotechnology. In particular, the observation of dynamical processes associated with DNA nanoassemblies—e.g., molecular walkers and machines—requires imaging techniques that are both fast and allow observation under native conditions. Here single-molecule fluorescence techniques and high-speed AFM are beginning to play an increasingly important role. WIREs Nanomed Nanobiotechnol 2012, 4:66–81. doi: 10.1002/wnan.173

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