Spectral Signal-to-Noise Ratio and Resolution Assessment of 3D Reconstructions

Measuring the quality of three-dimensional (3D) reconstructed biological macromolecules by transmission electron microscopy is still an open problem. In this article, we extend the applicability of the spectral signal-to-noise ratio (SSNR) to the evaluation of 3D volumes reconstructed with any reconstruction algorithm. The basis of the method is to measure the consistency between the data and a corresponding set of reprojections computed for the reconstructed 3D map. The idiosyncrasies of the reconstruction algorithm are taken explicitly into account by performing a noise-only reconstruction. This results in the definition of a 3D SSNR which provides an objective indicator of the quality of the 3D reconstruction. Furthermore, the information to build the SSNR can be used to produce a volumetric SSNR (VSSNR). Our method overcomes the need to divide the data set in two. It also provides a direct measure of the performance of the reconstruction algorithm itself; this latter information is typically not available with the standard resolution methods which are primarily focused on reproducibility alone

Status and Prospects of ZnO-Based Resistive Switching Memory Devices

In the advancement of the semiconductor device technology, ZnO could be a prospective alternative than the other metal oxides for its versatility and huge applications in different aspects. In this review, a thorough overview on ZnO for the application of resistive switching memory (RRAM) devices has been conducted. Various efforts that have been made to investigate and modulate the switching characteristics of ZnO-based switching memory devices are discussed. The use of ZnO layer in different structure, the different types of filament formation, and the different types of switching including complementary switching are reported. By considering the huge interest of transparent devices, this review gives the concrete overview of the present status and prospects of transparent RRAM devices based on ZnO. ZnO-based RRAM can be used for flexible memory devices, which is also covered here. Another challenge in ZnO-based RRAM is that the realization of ultra-thin and low power devices. Nevertheless, ZnO not only offers decent memory properties but also has a unique potential to be used as multifunctional nonvolatile memory devices. The impact of electrode materials, metal doping, stack structures, transparency, and flexibility on resistive switching properties and switching parameters of ZnO-based resistive switching memory devices are briefly compared. This review also covers the different nanostructured-based emerging resistive switching memory devices for low power scalable devices. It may give a valuable insight on developing ZnO-based RRAM and also should encourage researchers to overcome the challenges

Structure determination of two pore forming toxins by cryo-negative staining and single particle three-Dimensional reconstruction

Abtract: Cryo-electron microscopy of biological unordered and non aggregated molecules is a powerful approach for macromolecular assemblies, which are often too large and flexible to be studied by X-ray crystallography. Low temperature that preserves the native hydrated macromolecular structure confers to this technique the power to study conformational changes. Electron micrographs are very noisy and show low intrinsic contrast. Although there is no limits to the mass of the particles that can be analysed, experience place a lower size cutoff of ~250 kDa molecular weight. In order to go beyond these limits, the cryo-negative staining has been developped in our laboratory. The presence of a stain (molybdate amonium) makes possible to vizualize the particles with a enhanced SNR while keeping the specimen in a good state of preservation and reducing electron-beam sensity of vitrified specimen. In the present work, this method allowed us the study of two different pore forming toxins (PFTs), the Helicobacter Pylori vacuolating toxin,'VacA' unrelated to any other proteins, and a mutant form of the Aeromonas Hydrophila toxin, aerolysin (Y221G). Image analysis and 3D reconstruction of the wild type toxin VacA revealed that the monomer assembled into a number of different oligomers, all of which keeping elements of remarkable mobility. The structural comparaison of the wild type form of VacA and a mutant form where a unique hydrophobic region located near its amino terminus was deleted helped us to localize this hydrophobic domain whithin the VacA oligomeric structure and to provide insight into how this domain contributes to the formation of membrane channels by VacA. A high resolution model of a mutant form of aerolysin (Y221G), where a single point mutation has converted a normally membrane-embeded toxin into a soluble complex was obtained by the combination of X-ray data with the electron microscopy map. This model helped us to understand the role of the tyrosine 221 in the exposition of the hydrophobic regions that form the membrane- inserted channel. Finally, the small protein (~200 kDa) dipeptidyl-peptidase IV was also any studied by this technique. Résumé La cryo-microscopie électronique des molécules biologiques désordonnées et désagrégées est une approche puissante pour l'étude structurale d'assemblages macromoléculaires trop volumineux pour être étudiés par cristallographie aux rayons X. La basse température à laquelle les spécimens sont observés préserve les macromolécules dans leur état natif et hydraté et confère à cette technique le pouvoir d'étudier les différents états conformationnels. Les images de microscopie électronique obtenues, sont bruitées et présentent un contraste faible limitant ainsi l'observation de particules de faible poids moléculaire d'environ 250 kDa. Afin de surpasser ces limites, la cryo-coloration négative a été développée dans notre laboratoire. La présence d'un colorant (le molybdate d'ammonium) permet la visualisation des particules avec un rapport signal-sur-bruit augmenté tout en préservant le spécimen et en le protégeant des dégâts du faisceau électronique. Dans ce travail, cette méthode nous a permis d'étudier deux toxines bactériennes différentes formant des pores membranaires, la toxine VacA d'Helicobacter pyïori apparentée à aucune autre toxine connue et une forme mutante de la toxine aérolysine d'Aeromonas Hydrophila (Y221G). Le traitement d'images et la reconstruction tri-dimensionnelle de la toxine VacA de type sauvage a permis de mettre en lumière l'assemblage des monomères en oligomères tout en conservant des éléments d'une remarquable mobilité. La structure de la forme mutante de VacA pour laquelle l'unique région hydrophobe a été tronquée est comparée avec celle de la forme de type sauvage Cette comparaison a mis en évidence la localisation de ce domaine hydrophobe à l'intérieur de la structure des oligomères ainsi que la contribution de ce domaine dans la formation de canaux membranaires par VacA. La toxine aérolysine (Y221G) est le résultat d'une mutation unique qui permet la conversion d'une toxine insérée dans une membrane en un complexe soluble. Un modèle à l'échelle atomique a été obtenu pour ce mutant en combinant la carte de microscopie électronique tri-dimensionnelle aux données de cristallographie aux rayons X. Ce modèle souligne le rôle de la tyrosine 221 dans l'exposition des régions hydrophobes qui forment le canal membranaire. Enfin, la dernière protéine étudiée, la dipeptidyl-peptidase (DPPIV) (~200 kDa) a démontré que la cryo-coloration négative permet l'étude structurale de protéines plus petites que celles qui ont été d'étudiées jusqu'ici

High resolution structural analysis of Helicobacter pylori VacA toxin oligomers by cryo-negative staining electron microscopy.

Helicobacter pylori secretes a vacuolating toxin (VacA) that can assemble into water-soluble oligomeric complexes and insert into membranes to form anion-selective channels. Previous studies have described multiple types of oligomeric VacA structures, including single-layered astral arrays, bilayered forms, and two-dimensional crystalline arrays. In the current study, vitrified VacA complexes were examined by cryo-negative staining electron microscopy, views of the different oligomeric structures in multiple orientations were classified and analyzed, and three-dimensional models of the bilayered forms of VacA were constructed with a resolution of about 19 angstroms. These bilayered forms of VacA have a "flower"-like structure, consisting of a central ring surrounded by symmetrically arranged peripheral "petals." Further structural insights were obtained by analyzing a mutant form of VacA (VacADelta6-27), which lacks a unique amino-terminal hydrophobic segment and is defective in the capacity to form membrane channels. Bilayered oligomeric complexes formed by wild-type VacA contained a visible density within the central ring, whereas bilayered complexes formed by VacADelta6-27 lacked this density. These results indicate that deletion of the VacA amino-terminal hydrophobic region causes a structural alteration in the central ring within VacA oligomers, and suggest that the central ring plays an important role in the process by which VacA forms membrane channels

Cryo-negative staining reduces electron-beam sensitivity of vitrified biological particles

Beam damage is the main resolution-limiting factor when biological particles are observed by cryoelectron microscopy in a thin vitrified solution film. Furthermore, the low contrast of the specimen frequently makes observation difficult and limits the possibility of image processing. Cryo-negative staining, in which the particles are vitrified in a thin layer of concentrated ammonium molybdate solution, makes it possible to visualize the particles with a much better signal-to-noise ratio (SNR) while keeping the specimen in a good state of preservation. We have observed the Escherichia coli GroEL chaperonin, prepared in a native vitrified solution and by cryo-negative staining after electron exposure from 1000 to 3000e(-)/nm(2). We have compared the resulting three-dimensional models obtained from these different conditions and have tested their fit with the atomic model of the protein subunit obtained from X-ray crystallography. It is found that, down to 1.5-nm resolution, the particles appear to be faithfully represented in the cryo-negatively stained preparation, but there is an approximately 10-fold increase of SNR compared with the native vitrified preparation. Furthermore, for the same range of irradiation and down to the same resolution, the particles seem unaffected by beam damage, whereas the damage is severe in the native vitrified particles

Spline-based image-to-volume registration for three-dimensional electron microscopy

This paper presents an algorithm based on a continuous framework for a posteriori angular and translational assignment in three-dimensional electron microscopy (3DEM) of single particles. Our algorithm can be used advantageously to refine the assignment of standard quantized-parameter methods by registering the images to a reference 3D particle model. We achieve the registration by employing a gradient-based iterative minimization of a least-squares measure of dissimilarity between an image and a projection of the volume in the Fourier transform (FT) domain. We compute the FT of the projection using the central-slice theorem (CST). To compute the gradient accurately, we take advantage of a cubic B-spline model of the data in the frequency domain. To improve the robustness of the algorithm, we weight the cost function in the FT domain and apply a "mixed" strategy for the assignment based on the minimum value of the cost function at registration for several different initializations. We validate our algorithm in a fully controlled simulation environment. We show that the mixed strategy improves the assignment accuracy; on our data, the quality of the angular and translational assignment was better than 2 voxel (i.e., 6.54&ANGS;). We also test the performance of our algorithm on real EM data. We conclude that our algorithm outperforms a standard projection-matching refinement in terms of both consistency of 3D reconstructions and spee

A multiresolution approach to orientation assignment in 3D electron microscopy of single particles.

Three-dimensional (3D) electron microscopy (3DEM) aims at the determination of the spatial distribution of the Coulomb potential of macromolecular complexes. The 3D reconstruction of a macromolecule using single-particle techniques involves thousands of 2D projections. One of the key parameters required to perform such a 3D reconstruction is the orientation of each projection image as well as its in-plane orientation. This information is unknown experimentally and must be determined using image-processing techniques. We propose the use of wavelets to match the experimental projections with those obtained from a reference 3D model. The wavelet decomposition of the projection images provides a framework for a multiscale matching algorithm in which speed and robustness to noise are gained. Furthermore, this multiresolution approach is combined with a novel orientation selection strategy. Results obtained from computer simulations as well as experimental data encourage the use of this approach