This protocol describes a two-color astigmatic imaging approach that enables direct 3D visualization of cargo transport trajectories relative to a super-resolved octagonal double-ring scaffold structure of the nuclear pore complex (NPC). Though astigmatism imaging is commonly achieved via a cylindrical lens, this protocol utilizes an adaptive optics (AO) system, which enables optimization of the astigmatism for the precision needs of the experiment as well as correction of the focal mismatch arising from chromatic aberrations in multi-color applications. With this approach, single particle spatial precision values in x, y, and z are typically 5-20 nm, and these depend on astigmatism, photon level and position in z. The method enables resolution of transport conduits through the ~60 nm diameter pore of NPCs by particle tracking on the millisecond timescale. The success of this approach is enabled by the high rigidity of fully active NPCs within the nuclear envelope of permeabilized cells. For a detailed application of this protocol, please refer to https://www.nature.com/articles/s41556-021-00815-6. The figure and table numbers in this protocol that are indicated with an “NCB” prefix (e.g., NCB Figure X) refer to the figures and table in this reference paper.