Approche problème inverse pour l’alignement de séries en tomographie électronique

International audienceIn the refining industry, morphological measurements of particles have become an essential part in the characterization catalyst supports. Through these parameters, one can infer the specific physicochemical properties of the studied materials. One of the main acquisition techniques is electron tomography (or nanotomography). 3D volumes are reconstructed from sets of projections from different angles made by a Transmission Electron Microscope (TEM). This technique provides a real three-dimensional information at the nanometric scale. A major issue in this method is the misalignment of the projections that contributes to the reconstruction. The current alignment techniques usually employ fiducial markers such as gold particles for a correct alignment of the images. When the use of markers is not possible, the correlation between adjacent projections is used to align them. However, this method sometimes fails. In this paper, we propose a new method based on the inverse problem approach where a certain criterion is minimized using a variant of the Nelder and Mead simplex algorithm. The proposed approach is composed of two steps. The first step consists of an initial alignment process, which relies on the minimization of a cost function based on robust statistics measuring the similarity of a projection to its previous projections in the series. It reduces strong shifts resulting from the acquisition between successive projections. In the second step, the pre-registered projections are used to initialize an iterative alignment-refinement process which alternates between (i) volume reconstructions and (ii) registrations of measured projections onto simulated projections computed from the volume reconstructed in (i). At the end of this process, we have a correct reconstruction of the volume, the projections being correctly aligned. Our method is tested on simulated data and shown to estimate accurately the translation, rotation and scale of arbitrary transforms. We have successfully tested our method with real projections of different catalyst supports.Dans le domaine du raffinage, les mesures morphologiques de particules sont devenues indispensables pour caractériser les supports de catalyseurs. A travers ces paramètres, on peut remonter aux spécificités physico-chimiques des matériaux étudiés. Une des techniques d’acquisition utilisées est la tomographie électronique (ou nanotomographie). Des volumes 3D sont reconstruits à partir de séries de projections sous différents angles obtenues par Microscopie Électronique en Transmission (MET). Cette technique permet d’obtenir une réelle information tridimensionnelle à l’échelle du nanomètre. Un problème majeur dans ce contexte est le mauvais alignement des projections qui contribuent à la reconstruction. Les techniques d’alignement actuelles emploient habituellement des marqueurs de réference tels que des nanoparticules d’or pour un alignement correct des images. Lorsque l’utilisation de marqueurs n’est pas possible, l’alignement de projections adjacentes est obtenu par corrélation entre ces projections. Cependant, cette méthode échoue parfois. Dans cet article, nous proposons une nouvelle méthode basée sur une approche de type problème inverse où un certain critère est minimisé en utilisant une variante de l’algorithme de Nelder et Mead, qui exploite le concept de simplexe. Elle est composéé de deux étapes. La première étape consiste en un processus d’alignement initial s’appuyant sur la minimisation d’une fonction de coût basée sur des statistiques robustes, mesurant la similarité entre une projection et les projections précédentes de la série. Elle vise à réduire les forts déplacements, résultant de l’acquisition entre les projections successives. Dans la seconde étape, les projections pré-recalées sont employées pour initialiser un processus itératif et alterné d’alignement et reconstruction, minimisant alternativement une fonction de coût basée sur la reconstruction du volume et une fonction basée sur l’alignement d’une projection avec sa version simulée obtenue à partir du volume reconstruit. A la fin de ce processus, nous obtenons une reconstruction correcte du volume, les projections étant correctement alignées. Notre méthode a été testée sur des données simulées et prouve qu’elle récupère d’une manière précise les changements dans les paramètres de translation, rotation et mise à l’échelle. Nous avons testé avec succès notre méthode pour les projections réelles de différents supports de catalyseur

Improving data quality for 3D electron diffraction (3D ED) by Gatan Image Filter and a new crystal tracking method

3D ED is an effective technique to determine the structures of submicron- or nano-sized crystals. In this paper, we implemented energy-filtered 3D ED using a Gatan Energy Filter (GIF) in both selected area electron diffraction mode and micro/nanoprobe mode. We explained the setup in detail, which improves the accessibility of energy-filtered 3D ED experiments as more electron microscopes are equipped with a GIF than an in-column filter. We also proposed a crystal tracking method in STEM mode using live HAADF image stream. This method enables us to collect energy-filtered 3D ED datasets in STEM mode with a larger tilt range without foregoing any frames. In order to compare the differences between energy-filtered 3D ED and normal 3D ED data, three crystalline samples have been studied in detail. We observed that the final R1 will improve 20% to 30% for energy-filtered datasets compared with unfiltered datasets and the structure became more reasonable. We also discussed the possible reasons that lead to the improvement

Quantitatively Imaging Chromosomes by Correlated Cryo-Fluorescence and Soft X-Ray Tomographies

AbstractSoft x-ray tomography (SXT) is increasingly being recognized as a valuable method for visualizing and quantifying the ultrastructure of cryopreserved cells. Here, we describe the combination of SXT with cryogenic confocal fluorescence tomography (CFT). This correlative approach allows the incorporation of molecular localization data, with isotropic precision, into high-resolution three-dimensional (3-D) SXT reconstructions of the cell. CFT data are acquired first using a cryogenically adapted confocal light microscope in which the specimen is coupled to a high numerical aperture objective lens by an immersion fluid. The specimen is then cryo-transferred to a soft x-ray microscope (SXM) for SXT data acquisition. Fiducial markers visible in both types of data act as common landmarks, enabling accurate coalignment of the two complementary tomographic reconstructions. We used this method to identify the inactive X chromosome (Xi) in female v-abl transformed thymic lymphoma cells by localizing enhanced green fluorescent protein-labeled macroH2A with CFT. The molecular localization data were used to guide segmentation of Xi in the SXT reconstructions, allowing characterization of the Xi topological arrangement in near-native state cells. Xi was seen to adopt a number of different topologies with no particular arrangement being dominant

Structural Basis of Flagellar Filament Asymmetry and Supercoil Templating by Leptospira Sheath Factors

The spirochete family of bacteria, including pathogens such as Borrelia burgdorferi (Lyme disease), Treponema pallidum (syphilis), and Leptospira interrogans (leptospirosis), generates a unique, corkscrew-like form of motility that is crucial for pathogenicity. This motility is driven by flagella that are periplasmic rather than extracellular in nature and are composed of a FlaB core and a FlaA sheath. Purified Leptospira flagellar filaments form tight coils and contain the coiling proteins FcpA and FcpB, in addition to two FlaA isoforms. Loss of either the FlaA or Fcp proteins results in straighter flagella and bacteria that are non-motile and non-pathogenic. I have used cryo-electron microscopy to solve the structure of three Leptospira flagellar mutants to near-atomic resolution. First, I resolved the structure of an fcpB- mutant, allowing for identification of the FcpB density in the sheath region of the asymmetric wild-type structure. I also developed a method to quantify the curvature of purified flagellar filaments, allowing us to investigate how the flagellar forms are affected by various mutations. Next, I determined the structure of a fcpA- mutant, and found that the FlaA sheath components strictly localize to the inner curvature. We also discovered a previously uncharacterized flagellar sheath protein, which we have named FlaAP (FlaA-associated protein). We found that these sheath proteins interact with the core through interactions with glycans, and that the FlaB core appears to have ten ‘L’ protofilaments and one ‘R’ protofilament, with the ‘R’ protofilament directly underlying the sheath proteins and disrupting the helical symmetry of the core. Third, I solved the structure of an flaA2- mutant. In doing so, I found that whereas the Fcp coiling factors bind solely to the outer curvature of the wild-type filament, forming an extensive helical lattice, this Fcp lattice extends symmetrically around the entire FlaB core when the FlaA sheath factors are missing. These three mutant flagellar structures provide crucial insight into the role of the flagellar sheath factors and highlight the importance of the supercoiled wild-type form for motility and virulence. This work has implications for other spirochetes as well as other flagellated bacteria, where structural work has mainly been focused on flagellar filaments rendered straight by specific mutations, instead of the naturally occurring supercoiled forms as we have described here