ClusterAlign: A fiducial tracking and tilt series alignment tool for thick sample tomography

Abstract

Abstract Thick specimens, as encountered in cryo-scanning transmission electron tomography, offer special challenges to conventional reconstruction workflows. The visibility of features, including gold nanoparticles introduced as fiducial markers, varies strongly through the tilt series. As a result, tedious manual refinement may be required in order to produce a successful alignment. Information from highly tilted views must often be excluded to the detriment of axial resolution in the reconstruction. We introduce here an approach to tilt series alignment based on identification of fiducial particle clusters that transform coherently in rotation, essentially those that lie at similar depth. Clusters are identified by comparison of tilted views with a single untilted reference, rather than with adjacent tilts. The software, called ClusterAlign, proves robust to poor signal to noise ratio and varying visibility of the individual fiducials and is successful in carrying the alignment to the ends of the tilt series where other methods tend to fail. ClusterAlign may be used to generate a list of tracked fiducials, to align a tilt series, or to perform a complete 3D reconstruction. Tools to evaluate alignment error by projection matching are included. Execution involves no manual intervention, and adherence to standard file formats facilitates an interface with other software, particularly IMOD/etomo, tomo3d, and tomoalign.