AbstractFull-field transmission X-ray microscopy (TXM) is a well established technique, available at various synchrotron beamlines around the world as well as by laboratory benchtop devices. One of the major TXM challenges, due to its nanometre-scale resolution, is the overall instrument stability during the acquisition of the series of tomographic projections. The ability to correct for vertical and horizontal distortions of each projection image during acquisition is necessary in order to achieve the effective 3D spatial resolution. The effectiveness of such an image alignment is also heavily influenced by the absorption properties and strong contrast of specific features in the scanned sample. Here it is shown that nanoporous gold (NPG) can be used as an ideal 3D test pattern for evaluating and optimizing the performance of a TXM instrument for hard X-rays at a synchrotron beamline. Unique features of NPG, such as hierarchical structures at multiple length scales and high absorbing capabilities, makes it an ideal choice for characterization, which involves a combination of a rapid-alignment algorithm applied on the acquired projections followed by the extraction of a set of both 2D- and 3D-descriptive image parameters. This protocol can be used for comparing the efficiency of TXM instruments at different synchrotron beamlines in the world or benchtop devices, based on a reference library of scanned NPG samples, containing information about the estimated horizontal and vertical alignment values, 2D qualitative parameters and quantitative 3D parameters. The possibility to tailor the ligament sizes of NPG to match the achievable resolution in combination with the high electron density of gold makes NPG an ideal 3D test pattern for evaluating the status and performance of a given synchrotron-based or benchtop-based TXM setup.