Novel computational methods for in vitro and in situ cryo-electron microscopy

Over the past decade, advances in microscope hardware and image data processing algorithms have made cryo-electron microscopy (cryo-EM) a dominant technique for protein structure determination. Near-atomic resolution can now be obtained for many challenging in vitro samples using single-particle analysis (SPA), while sub-tomogram averaging (STA) can obtain sub-nanometer resolution for large protein complexes in a crowded cellular environment. Reaching high resolution requires large amounts of im-age data. Modern transmission electron microscopes (TEMs) automate the acquisition process and can acquire thousands of micrographs or hundreds of tomographic tilt se-ries over several days without intervention. In a first step, the data must be pre-processed: Micrographs acquired as movies are cor-rected for stage and beam-induced motion. For tilt series, additional alignment of all micrographs in 3D is performed using gold- or patch-based fiducials. Parameters of the contrast-transfer function (CTF) are estimated to enable its reversal during SPA refine-ment. Finally, individual protein particles must be located and extracted from the aligned micrographs. Current pre-processing algorithms, especially those for particle picking, are not robust enough to enable fully unsupervised operation. Thus, pre-processing is start-ed after data collection, and takes several days due to the amount of supervision re-quired. Pre-processing the data in parallel to acquisition with more robust algorithms would save time and allow to discover bad samples and microscope settings early on. Warp is a new software for cryo-EM data pre-processing. It implements new algorithms for motion correction, CTF estimation, tomogram reconstruction, as well as deep learn-ing-based approaches to particle picking and image denoising. The algorithms are more accurate and robust, enabling unsupervised operation. Warp integrates all pre-processing steps into a pipeline that is executed on-the-fly during data collection. Inte-grated with SPA tools, the pipeline can produce 2D and 3D classes less than an hour into data collection for favorable samples. Here I describe the implementation of the new algorithms, and evaluate them on various movie and tilt series data sets. I show that un-supervised pre-processing of a tilted influenza hemagglutinin trimer sample with Warp and refinement in cryoSPARC can improve previously published resolution from 3.9 Å to 3.2 Å. Warp’s algorithms operate in a reference-free manner to improve the image resolution at the pre-processing stage when no high-resolution maps are available for the particles yet. Once 3D maps have been refined, they can be used to go back to the raw data and perform reference-based refinement of sample motion and CTF in movies and tilt series. M is a new tool I developed to solve this task in a multi-particle framework. Instead of following the SPA assumption that every particle is single and independent, M models all particles in a field of view as parts of a large, physically connected multi-particle system. This allows M to optimize hyper-parameters of the system, such as sample motion and deformation, or higher-order aberrations in the CTF. Because M models these effects accurately and optimizes all hyper-parameters simultaneously with particle alignments, it can surpass previous reference-based frame and tilt series alignment tools. Here I de-scribe the implementation of M, evaluate it on several data sets, and demonstrate that the new algorithms achieve equally high resolution with movie and tilt series data of the same sample. Most strikingly, the combination of Warp, RELION and M can resolve 70S ribosomes bound to an antibiotic at 3.5 Å inside vitrified Mycoplasma pneumoniae cells, marking a major advance in resolution for in situ imaging

Morphological characterization of a polymeric microfiltration membrane by synchrotron radiation computed microtomography

Most commercial polymeric membranes are prepared by phase inversion. The performance of the membranes depends greatly on the morphology of the porous structure formed during the different steps of this process. Researchers in this field have found it extremely difficult to foresee how a change in the composition of the polymer solution will affect pore formation without a set of methods designed to yield detailed knowledge of the morphological structure. This paper reports the new potential associated with X-Ray synchrotron microtomography to characterize the 3D structure of a PvDF hollow fibre microfiltration membrane prepared by phase inversion. 3D morphological data obtained from the ID19 line at the ESRF are presented. The membrane actually appears as a complex three-dimensional bi-continuum of interconnected pores. Within the hollow fibre structure, different regions with various thicknesses and pore size distributions have been identified and well characterized. Transversal views show the anisotropic finger-like structure of pores, while longitudinal sections reveal a honeycomb structure which resembles the structure of highly concentrated water in oil emulsion or dispersion. This typical structure might be obtained during the phase inversion process. How the phase inversion process may result in these morphologies is finally discussed

Quantifying the hydroxyapatite orientation near the ossification front in a piglet femoral condyle using X-ray diffraction tensor tomography

While a detailed knowledge of the hierarchical structure and morphology of the extracellular matrix is considered crucial for understanding the physiological and mechanical properties of bone and cartilage, the orientation of collagen fibres and carbonated hydroxyapatite (HA) crystallites remains a debated topic. Conventional microscopy techniques for orientational imaging require destructive sample sectioning, which both precludes further studies of the intact sample and potentially changes the microstructure. In this work, we use X-ray diffraction tensor tomography to image non-destructively in 3D the HA orientation in a medial femoral condyle of a piglet. By exploiting the anisotropic HA diffraction signal, 3D maps showing systematic local variations of the HA crystallite orientation in the growing subchondral bone and in the adjacent mineralized growth cartilage are obtained. Orientation maps of HA crystallites over a large field of view (~ 3 7 3 7 3 mm3) close to the ossification (bone-growth) front are compared with high-resolution X-ray propagation phase-contrast computed tomography images. The HA crystallites are found to predominantly orient with their crystallite c-axis directed towards the ossification front. Distinct patterns of HA preferred orientation are found in the vicinity of cartilage canals protruding from the subchondral bone. The demonstrated ability of retrieving 3D orientation maps of bone-cartilage structures is expected to give a better understanding of the physiological properties of bones, including their propensity for bone-cartilage diseases

Use of seismic refraction in determining rock mass anisotropy

Seismic refraction is a popular method used by geological/geotechnical engineers to understand subsurface conditions. This method along with information collected from borings produce a realistic image of the ground beneath us. The goal of this study is to define and test a set of procedures to use seismic refraction alone to create high quality images of subsurface conditions. The focus is on areas with several meters of soil overlain by bedrock. The application of these procedures will minimize construction costs by eliminating the need for multiple boreholes. The results show that two important criteria must be met for successful application of the method: a bedrock outcrop near the survey area and an area wide enough for geophone spacing to reach the bedrock. Seismic profiles are to be laid radially in an orthogonal position preferably intersecting in the middle or in a quarter position but not at the end. Single channel geode is used for data acquisition and Rayfract® for data processing. Surfer® and Voxler® are used for graphical representations. Seismic refraction procedures are tested at two different sites: a road cut and an abandoned quarry. In one case (road cut) analysis did not show any anisotropy which resulted from failure to meet the second criterion (geophone spacing). Analysis from second area (quarry) exhibited a clear anisotropic nature of bedrock confirmed by observations on the exposed outcrop in the quarry. This is a qualitative study that can predict the orientation of major joint sets if the above criteria have been met

Orientational mapping of minerals in Pierre shale using X-ray diffraction tensor tomography

Shales have a complex mineralogy with structural features spanning several length scales, making them notoriously difficult to fully understand. Conventional attenuation-based X-ray computed tomography (CT) measures density differences, which, owing to the heterogeneity and sub-resolution features in shales, makes reliable interpretation of shale images a challenging task. CT based on X-ray diffraction (XRD-CT), rather than intensity attenuation, is becoming a well established technique for non-destructive 3D imaging, and is especially suited for heterogeneous and hierarchical materials. XRD patterns contain information about the mineral crystal structure, and crucially also crystallite orientation. Here, we report on the use of orientational imaging using XRD-CT to study crystallite-orientation distributions in a sample of Pierre shale. Diffraction-contrast CT data for a shale sample measured with its bedding-plane normal aligned parallel to a single tomographic axis perpendicular to the incoming X-ray beam are discussed, and the spatial density and orientation distribution of clay minerals in the sample are described. Finally, the scattering properties of highly attenuating inclusions in the shale bulk are studied, which are identified to contain pyrite and clinochlore. A path forward is then outlined for systematically improving the structural description of shales.publishedVersio