Hydration layers are ubiquitous in life and technology. Hence, interfacial aqueous layers have a central role in a wide range of phenomena from materials science to molecular and cell biology. A complete understanding of those processes requires, among other things, the development of very-sensitive and high-resolution instruments. Three-dimensional atomic force microscopy (3D-AFM) represents the latest and most successful attempt to generate atomically resolved three-dimensional images of solid-liquid interfaces. This review provides an overview of the 3D-AFM operating principles and its underlying physics. We illustrate and explain the capability of the instrument to resolve atomic defects on crystalline surfaces immersed in liquid. We also illustrate some of its applications to imaging the hydration structures on DNA or proteins. In the last section, we discuss some perspectives on emerging applications in materials science and molecular biology.