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Atomic force microscopy (AFM) is a powerful tool for imaging individual bio‐macromolecules [1, 2]. In particular, its range of operation has been suitable for the morphological analysis of nucleic acid molecules ranging from a single DNA fragment (several tens of nanometers) to a large assembly of DNA nanostructures (several hundred nanometers to a few micrometers). Initial attempts to use the instrument for imaging DNA molecules date back to the early days of AFM. Throughout the late 1980s to the 1990s, it was notably applied to observe double‐stranded DNA [3–6], and new preparation methods for nucleic acid specimens amenable to AFM imaging were developed [7, 8]. These achievements greatly encouraged investigation of various DNA–protein assemblies [9, 10] and artificially designed DNA nanostructures [11]. In particular, in the field of structural nucleic acid nanotechnology, AFM has been routinely used to visualize various types of artificial DNA nanostructures, such as DNA tile motifs [11], DNA origamis [12], and DNA bricks [13], and it has become an almost indispensable tool.

Compared with electron microscopy, AFM offers the advantage of being able to measure the surface topography of specimens without the need for chemical staining or fixation. Furthermore, its capability to scan samples in an aqueous solution offers the potential to directly image dynamic movements of biomolecules at nanoscale resolution under user‐defined buffer solutions; however, this type of application had been limited for a long time due to the inherently slow scanning rate of conventional AFM, which takes several minutes to produce a single image. It should be mentioned that although the conventional instrument cannot capture rapid molecular motions, dynamic events involving structural changes of the molecule of interest can often be interpreted even with the slow scanning rate by comparing “before” and “after” images. However, it is clearly more ideal to see a single molecule in action or dynamic events involving numbers of molecules in the same imaging area in real time. High‐speed AFM (HS‐AFM) introduced by Ando realized a scan rate of >1 frame/second (fps) [14], and it has been applied to unravel structural–function relationships of a variety of biological molecules, including proteins, protein–protein complexes, and protein–nucleic acid complexes [15–17]. HS‐AFM is now also employed in the field of structural DNA nanotechnology for the development of DNA nanomachines and studies of dynamic processes of artificial self‐assembly systems made up of DNA nanostructures. This chapter focuses on how the time‐lapse AFM technique has been utilized to study structural changes of DNA origami nanomachines, dynamic processes of two‐dimensional (2D) DNA origami lattice self‐assembly, and morphological changes of other DNA origami‐based dynamic systems.