10. Substrate Surfaces

The substrate surfaces onto which samples are placed play an essential role in successful dynamic imaging by HS-AFM. In contrast to single-molecule fluorescence microscopy, for HS-AFM, the surface roughness should be small enough to identify molecules of interest, which typically have single-nanometer dimensions. Moreover, because AFM can visualize the sample only from one direction perpendicular to the substrate surface, sample molecules sometimes have to be attached to the surface in a specific orientation so that their characteristic structural features and portions of interest of the molecules can be visualized. These requirements are common in both static and dynamic AFM imaging. However, in dynamic AFM imaging, sample molecules should not strongly interact with a surface, which could interfere with their function. Nevertheless, when the interaction is too weak, the molecules move too fast on the surface to be clearly imaged even with HS-AFM.