Electron tomography has become an essential tool for three-dimensional (3D) characterization of nanomaterials. In recent years, advances have been made in specimen preparation and mounting, acquisition geometries, and reconstruction algorithms. All of these components work together to optimize the resolution and clarity of an electron tomogram. However, one important component of the data-processing has received less attention: the 2D tilt series alignment. This is challenging for a number of reasons, namely because the nature of the data sets and the need to be coherently aligned over the full range of angles. An inaccurate alignment may be difficult to identify, yet can significantly limit the final 3D resolution. In this work, we present an improved center-of-mass alignment model that allows us to overcome discrepancies from unwanted objects that enter the imaging area throughout the tilt series. In particular, we develop an approach to overcome changes in the total mass upon rotation of the imaging area. We apply our approach to accurately recover small Pt nanoparticles embedded in a zeolite that may otherwise go undetected both in the 2D microscopy images and the 3D reconstruction. In addition to this, we highlight the particular effectiveness of the compressed sensing methods with this data set.

Combining the biological specificity of fluorescence microscopy with topographical features revealed by atomic force microscopy (AFM) provides new insights into cell biology. However, the lack of systematic alignment capabilities especially in scanning-tip AFM has limited the combined application approach as AFM drift leads to increasing image mismatch over time. We present an alignment correction method using the cantilever tip as a reference landmark. Since the precise tip position is known in both the fluorescence and AFM images, exact re-alignment becomes possible. We used beads to demonstrate the validity of the method in a complex artificial sample. We then extended this method to biological samples to depict membrane structures in fixed and living human fibroblasts. We were able to map nanoscale membrane structures, such as clathrin-coated pits, to their respective fluorescent spots. Reliable alignment between fluorescence signals and topographic structures opens possibilities to assess key biological processes at the cell surface such as endocytosis and exocytosis.