The invention of 3D atomic force microscopy (3D-AFM) has enabled visualizing subnanoscale 3D hydration structures. Meanwhile, its applications to imaging flexible molecular chains have started to be experimentally explored. However, the validity and principle of such imaging have yet to be clarified by comparing experiments and simulations or cross-observations with an alternative technique. Such studies are impeded by the lack of an appropriate model. Here, this difficulty is overcome by fabricating 3D carbon nanotube (CNT) structures flexible enough for 3D-AFM, large enough for scanning electron microscopy (SEM), and simple enough for simulations. SEM and 3D-AFM observations of the same model provide unambiguous evidence to support the possibility of imaging overlapped nanostructures, such as suspended CNT and underlying platinum (Pt) nanodots. Langevin dynamics simulations of such 3D-AFM imaging clarify the imaging mechanism, where the flexible CNT is laterally displaced to allow the AFM probe access to the underlying structures. These results consistently show that 3D-AFM images are affected by the friction between the CNT and AFM nanoprobe, yet it can be significantly suppressed by oscillating the cantilever. This study reinforces the theoretical basis of 3D-AFM for imaging various 3D self-organizing systems in diverse fields, from life sciences to interface sciences.|This work marks the first comprehensive explanation of the 3D atomic force micrscopy (3D-AFM) imaging mechanism for flexible structures. Investigating a 3D model structure featuring suspended carbon nanotube (CNT) provides valuable insights into measuring flexible structures with minimal disturbance. It also presents direct evidence that 3D-AFM is capable of visualizing vertically overlapped 3D nanostructures. image