AreTomo, an abbreviation for Alignment and Reconstruction for Electron Tomography, is a GPU accelerated software package that fully automates motion-corrected marker-free tomographic alignment and... Show moreAreTomo, an abbreviation for Alignment and Reconstruction for Electron Tomography, is a GPU accelerated software package that fully automates motion-corrected marker-free tomographic alignment and reconstruction in a single package. By correcting in-plane rotation, translation, and importantly, the local motion resulting from beam-induced motion from tilt to tilt, AreTomo can produce tomograms with sufficient accuracy to be directly used for subtomogram averaging. Another major application is the on-the-fly reconstruction of tomograms in parallel with tilt series collection to provide users with real-time feedback of sample quality allowing users to make any necessary adjustments of collection parameters. Here, the multiple alignment algorithms implemented in AreTomo are described and the local motions measured on a typical tilt series are analyzed. The residual local motion after correction for global motion was found in the range of +/- 80 angstrom, indicating that the accurate correction of local motion is critical for high-resolution cryo-electron tomography (cryoET). Show less
The research in this thesis is focused on tomographic reconstruction based on two imaging modalities in electron microscopy. The first modality is high angle annular dark field scanning... Show moreThe research in this thesis is focused on tomographic reconstruction based on two imaging modalities in electron microscopy. The first modality is high angle annular dark field scanning transmission microscopy (HAADF-STEM), and the second modality is energy-dispersive X-ray spectroscopy (EDS). In this Ph.D. thesis, we propose several approaches to pave the way for HAADF- STEM + EDS tomography: (1) the HAADF-EDS bimodal tomographic reconstruction technique, which is based on jointly modeling the consistency of the two imaging modalities; (2) TNV-regularized joined reconstruction which allows to incorporate the prior knowledge that common edges exist in the reconstructions from HAADF and EDS data respectively; (3) a set of algorithmic recipes to tailor various reconstruction algorithms for given experimental conditions and sample properties; (4) an algorithm for automatically correcting the nonlinear damping effects in HAADF-STEM tomographic data. Show less
Sharp, T.H.; Faas, F.G.A.; Koster, A.J.; Gros, P. 2017
Positive-stranded RNA viruses replicate in the cytoplasm of host cells and their replication complexes are associated with modified cell membranes. We investigated the structure of the nidovirus... Show morePositive-stranded RNA viruses replicate in the cytoplasm of host cells and their replication complexes are associated with modified cell membranes. We investigated the structure of the nidovirus-induced membrane modifications and found that nidoviruses transform the endoplasmic reticulum into a reticulovesicular network (RVN) that integrates numerous inter-connected double-membrane vesicles (DMVs), and, in the case of coronaviruses, __vesicle packets__ and convoluted membranes (CM). The membranes of DMVs and CM labeled abundantly for replicase proteins, whereas the DMV interior contained the double-stranded RNA and therefore is the presumed site of viral RNA synthesis. Electron tomography, semi-permeabilization and nuclease digestion experiments suggested that the DMV interior is inaccessible from the cytosol, implying that double-stranded RNA is compartmentalized by membranes. Furthermore, brefeldin A treatment showed that early secretory pathway proteins do not play a direct role in RVN morphogenesis or functionality of the SARS-CoV replication complex. Continued translation was not an absolute requirement for viral RNA synthesis, however, was necessary for RVN development thus suggesting a link between accumulation of replicase proteins and RVN formation. Together with biochemical studies of the viral enzyme complex, our ultrastructural description of the RVN aids to further dissect the early stages of the nidovirus infection and virus-host interactions. Show less
