This Perspective summarizes the recent advances and perspective in three-dimensional (3D) imaging techniques and their applications to polymer nanostructures, e.g., microphase-separated structures of block copolymers. We place particular emphasis on the method of transmission electron microtomography (TEMT). As a result of some recent developments in TEMT, it is now possible to obtain truly quantitative 3D images of polymer nanostructures with subnanometer resolution. The introduction of scanning optics in TEMT has made it possible to obtain large volumes of 3D data from a micrometer thick polymer specimens using conventional electron microscopes at relatively low acceleration voltage, 200 kV. Thus, TEMT covers structures over a wide range of thicknesses from a few nanometers to several hundred nanometers, which corresponds to quite an important spatial range for hierarchical polymer nanostructures. TEMT provides clear 3D images and a wide range of new structural information, which cannot be obtained using other methods, e.g., conventional microscopy or scattering methods, can be directly evaluated from the 3D volume data. In addition, when combined with other characterization methods, e.g., scattering and computer simulations, TEMT can yield even better results. The single chain conformation of block copolymers inside microdomains may be virtually visualized by TEMT. TEMT is a versatile technique that is not only restricted to polymer applications but can also be used as a powerful characterization tool in energy applications, e.g., fuel cells, etc. copy; 2010 American Chemical Society.