Electron Image Reconstruction Research Articles

Where bone meets implant: the characterization of nano-osseointegration

The recent application of electron tomography to the study of biomaterial interfaces with bone has brought about an awareness of nano-osseointegration and, to a further extent, demanded increasingly advanced characterization methodologies. In this study, nanoscale osseointegration has been studied via laser-modified titanium implants. The micro- and nano-structured implants were placed in the proximal tibia of New Zealand white rabbits for six months. High-resolution transmission electron microscopy (HRTEM), analytical microscopy, including energy dispersive X-ray spectroscopy (EDXS) and energy-filtered TEM (EFTEM), as well as electron tomography studies were used to investigate the degree of nano-osseointegration in two- and three-dimensions. HRTEM indicated the laser-modified surface encouraged the formation of crystalline hydroxyapatite in the immediate vicinity of the implant. Analytical studies suggested the presence of a functionally graded interface at the implant surface, characterized by the gradual intermixing of bone with oxide layer. Yet, the most compelling of techniques, which enabled straightforward visualization of nano-osseointegration, proved to be segmentation of electron tomographic reconstructions, where thresholding techniques identified bone penetrating into the nanoscale roughened surface features of laser-modified titanium. Combining high-resolution, analytical and three-dimensional electron microscopy techniques has proven to encourage identification and understanding of nano-osseointegration.

Electron Microscopic Analysis of a Spherical Mitochondrial Structure

Mitochondria undergo dynamic structural alterations to meet changing needs and to maintain homeostasis. We report here a novel mitochondrial structure. Conventional transmission electron microscopic examination of murine embryonic fibroblasts treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, found that more than half of the mitochondria presented a ring-shaped or C-shaped morphology. Many of these mitochondria seemed to have engulfed various cytosolic components. Serial sections through individual mitochondria indicated that they formed a ball-like structure with an internal lumen surrounded by the membranes and containing cytosolic materials. Notably, the lumen was connected to the external cytoplasm through a small opening. Electron tomographic reconstruction of the mitochondrial spheroids demonstrated the membrane topology and confirmed the vesicular configuration of this mitochondrial structure. The outside periphery and the lumen were defined by the outer membranes, which were lined with the inner membranes. Matrix and cristae were retained but distributed unevenly with less being kept near the luminal opening. Mitochondrial spheroids seem to form in response to oxidative mitochondrial damage independently of mitophagy. The structural features of the mitochondrial spheroids thus represent a novel mitochondrial dynamics.

A new approach to the investigation of nanoparticles: Electron tomography with compressed sensing

The principal purpose of this contribution is to illustrate the potential of compressed sensing electron tomography for the characterisation of nanoparticulate materials that are vulnerable to electron beam damage. Not only is there growing interest in nanoparticles of organic materials in medical and allied contexts, there is also the need to investigate nanoparticles and nanoclusters of metals supported on biological macromolecular entities in the context of drug delivery. A qualitative account of the principles of electron tomography is outlined with illustrations from the field of heterogeneous catalysis, where electron beam damage is less of an issue, and an appendix deals with more quantitative aspects of how compressed sensing promises to expand the range of samples that have hitherto been accessible to investigation.

Shape-Based Regularization of Electron Tomographic Reconstruction

We introduce a tomographic reconstruction method implemented using a shape-based regularization technique. Spatial models of known features in the structure being reconstructed are integrated into the reconstruction process as regularizers. Our regularization scheme is driven locally through shape information obtained from segmentation and compared with a known spatial model. We demonstrated our method on tomography data from digital phantoms, simulated data, and experimental electron tomography (ET) data of virus complexes. Our reconstruction showed reduced blurring and an improvement in the resolution of the reconstructed volume was also measured. This method also produced improved demarcation of spike boundaries in viral membranes when compared with popular techniques like weighted back projection and the algebraic reconstruction technique. Improved ET reconstructions will provide better structure elucidation and improved feature visualization, which can aid in solving key biological issues. Our method can also be generalized to other tomographic modalities.

Inhomogeneity of Nacre Lamellae on the Nanometer Length Scale

We present experimental evidence of inhomogeneous distribution of organic inclusions within individual aragonitic lamellae forming the nacre layer of mollusk shells. This nanoscale-inhomogeneity is visualized in the Perna canaliculus (green mussel) shells by the aid of high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) in the tomography mode. Electron tomography reconstructions of the three-dimensional distribution of intracrystalline organic inclusions, ranging in size between 2 to 25 nm, clearly reveal 50 nm wide depletion zones adjacent to the organic sheets between lamellae. The interrelation between the observed fine-scale inhomogeneity of nacre layer and its improved mechanical properties is discussed.

Pushing the resolution limits in cryo electron tomography of biological structures

Cryo electron tomography is a three-dimensional imaging technique that is suitable for imaging snapshots of the structural arrangements of biomolecular complexes and macromolecules, both in vitro and in the context of the cell. In terms of attainable resolution, cryo electron tomographic reconstructions now show resolvable details in the 5-10 nm range, connecting optical microscopy with molecular imaging techniques. In view of the current developments in super-resolution light microscopy and correlative light and electron microscopy, cryo electron tomography will be increasingly important in structural biology as a tool to bridge light microscopy with molecular imaging techniques like NMR, X-ray diffraction and single particle electron microscopy. In cell biology, one goal, often referred to as visual proteomics, is the molecular mapping of whole cells. To achieve this goal and link cryo electron tomography to these high-resolution techniques, increasing the attainable resolution to 2-5 nm is vital. Here, we provide an overview of technical factors that limit the resolution in cryo electron tomography and discuss how during data acquisition and image processing these can be optimized to attain the highest possible resolution. Also, existing resolution measurement approaches and current technological developments that potentially increase the resolution in cryo electron tomography are discussed.

High-performance blob-based iterative three-dimensional reconstruction in electron tomography using multi-GPUs

BackgroundThree-dimensional (3D) reconstruction in electron tomography (ET) has emerged as a leading technique to elucidate the molecular structures of complex biological specimens. Blob-based iterative methods are advantageous reconstruction methods for 3D reconstruction in ET, but demand huge computational costs. Multiple graphic processing units (multi-GPUs) offer an affordable platform to meet these demands. However, a synchronous communication scheme between multi-GPUs leads to idle GPU time, and a weighted matrix involved in iterative methods cannot be loaded into GPUs especially for large images due to the limited available memory of GPUs.ResultsIn this paper we propose a multilevel parallel strategy combined with an asynchronous communication scheme and a blob-ELLR data structure to efficiently perform blob-based iterative reconstructions on multi-GPUs. The asynchronous communication scheme is used to minimize the idle GPU time so as to asynchronously overlap communications with computations. The blob-ELLR data structure only needs nearly 1/16 of the storage space in comparison with ELLPACK-R (ELLR) data structure and yields significant acceleration.ConclusionsExperimental results indicate that the multilevel parallel scheme combined with the asynchronous communication scheme and the blob-ELLR data structure allows efficient implementations of 3D reconstruction in ET on multi-GPUs.

Automated tracing of filaments in 3D electron tomography reconstructions using Sculptor and Situs

The molecular graphics program Sculptor and the command-line suite Situs are software packages for the integration of biophysical data across spatial resolution scales. Herein, we provide an overview of recently developed tools relevant to cryo-electron tomography (cryo-ET), with an emphasis on functionality supported by Situs 2.7.1 and Sculptor 2.1.1. We describe a work flow for automatically segmenting filaments in cryo-ET maps including denoising, local normalization, feature detection, and tracing. Tomograms of cellular actin networks exhibit both cross-linked and bundled filament densities. Such filamentous regions in cryo-ET data sets can then be segmented using a stochastic template-based search, VolTrac. The approach combines a genetic algorithm and a bidirectional expansion with a tabu search strategy to localize and characterize filamentous regions. The automated filament segmentation by VolTrac compares well to a manual one performed by expert users, and it allows an efficient and reproducible analysis of large data sets. The software is free, open source, and can be used on Linux, Macintosh or Windows computers.

IPET and FETR: Experimental Approach for Studying Molecular Structure Dynamics by Cryo-Electron Tomography of a Single-Molecule Structure

The dynamic personalities and structural heterogeneity of proteins are essential for proper functioning. Structural determination of dynamic/heterogeneous proteins is limited by conventional approaches of X-ray and electron microscopy (EM) of single-particle reconstruction that require an average from thousands to millions different molecules. Cryo-electron tomography (cryoET) is an approach to determine three-dimensional (3D) reconstruction of a single and unique biological object such as bacteria and cells, by imaging the object from a series of tilting angles. However, cconventional reconstruction methods use large-size whole-micrographs that are limited by reconstruction resolution (lower than 20 Å), especially for small and low-symmetric molecule (<400 kDa). In this study, we demonstrated the adverse effects from image distortion and the measuring tilt-errors (including tilt-axis and tilt-angle errors) both play a major role in limiting the reconstruction resolution. Therefore, we developed a “focused electron tomography reconstruction” (FETR) algorithm to improve the resolution by decreasing the reconstructing image size so that it contains only a single-instance protein. FETR can tolerate certain levels of image-distortion and measuring tilt-errors, and can also precisely determine the translational parameters via an iterative refinement process that contains a series of automatically generated dynamic filters and masks. To describe this method, a set of simulated cryoET images was employed; to validate this approach, the real experimental images from negative-staining and cryoET were used. Since this approach can obtain the structure of a single-instance molecule/particle, we named it individual-particle electron tomography (IPET) as a new robust strategy/approach that does not require a pre-given initial model, class averaging of multiple molecules or an extended ordered lattice, but can tolerate small tilt-errors for high-resolution single “snapshot” molecule structure determination. Thus, FETR/IPET provides a completely new opportunity for a single-molecule structure determination, and could be used to study the dynamic character and equilibrium fluctuation of macromolecules.

Asymmetric Small Protein Structure Determination by Individual Particle Electron Tomography

Single-particle reconstruction of electron microscopy has been successfully used to determine high-resolution structure of proteins that are highly symmetric with a large macromolecular complex (molecular weight, MW > 200 kDa). However, for asymmetric small proteins (MW < 100 kDa), this approach still faces challenges to obtain intermediate resolution structure due to difficulties attaining a reliable initial model. To obtain the initial model, especially from an asymmetric small protein, we present a novel method by individual particle electron tomography (IPET). In this method, an individual protein particle was targeted and imaged from a serials of tilt-angles by electron tomography (ET) using our reported focused ET reconstruction (FETR) algorithm to reconstruct the three-dimensional (3D) structure from those tilt serial of images that were acquired from each instance of the molecule. To demonstrate the method, we imaged and reconstructed structure of small protein, cholesteryl ester transfer protein (CETP), whose molecular weight is only 53 kDa. The experimental sample was prepared by an optimized negative-staining protocol and was acquired under the near Scherzer-focus imaging condition. To investigate the reliability of the 3D density map reconstructed from single-instance particle, we statistically analyzed the map by comparing it with the crystal structure and the density map refined from this initial model, followed by the single-particle refinement procedure. Although Fourier shell cross-correlation (FSC) analysis shows the resolution of the tomographic reconstruction and single-particle reconstruction are both around 15 A. These results suggest this approach can be used to obtain the initial model for single-particle reconstruction, moreover, the similar quality and resolution obtained from the IPET and single-particle reconstruction suggest that our reported IPET and FETR reconstruction can be used directly to determine the single-instance molecule structure at intermediate resolution (1-2 nm).

Reconstruction and visualization of nanoparticle composites by transmission electron tomography

This paper examines the limits of transmission electron tomography reconstruction methods for a nanocomposite object composed of many closely packed nanoparticles. Two commonly used reconstruction methods in TEM tomography were examined and compared, and the sources of various artefacts were explored. Common visualization methods were investigated, and the resulting “interpretation artefacts” ( i.e., deviations from “actual” particle sizes and shapes arising from the visualization) were determined. Setting a known or estimated nanoparticle volume fraction as a criterion for thresholding does not in fact give a good visualization. Unexpected effects associated with common built-in image filtering methods were also found. Ultimately, this work set out to establish the common problems and pitfalls associated with electron beam tomographic reconstruction and visualization of samples consisting of closely spaced nanoparticles.

Performance improvements for iterative electron tomography reconstruction using graphics processing units (GPUs)

Iterative reconstruction algorithms are becoming increasingly important in electron tomography of biological samples. These algorithms, however, impose major computational demands. Parallelization must be employed to maintain acceptable running times. Graphics Processing Units (GPUs) have been demonstrated to be highly cost-effective for carrying out these computations with a high degree of parallelism. In a recent paper by Xu et al. (2010), a GPU implementation strategy was presented that obtains a speedup of an order of magnitude over a previously proposed GPU-based electron tomography implementation. In this technical note, we demonstrate that by making alternative design decisions in the GPU implementation, an additional speedup can be obtained, again of an order of magnitude. By carefully considering memory access locality when dividing the workload among blocks of threads, the GPU's cache is used more efficiently, making more effective use of the available memory bandwidth.

ATOM 1.0: A GPU Powered Package for Electron Tomography Reconstruction

ATOM 1.0: A GPU Powered Package for Electron Tomography Reconstruction

Three-dimensional reconstruction using an adaptive simultaneous algebraic reconstruction technique in electron tomography

Three-dimensional (3D) reconstruction of electron tomography (ET) has emerged as an important technique in analyzing structures of complex biological samples. However most of existing reconstruction methods are not suitable for extremely noisy and incomplete data conditions. We present an adaptive simultaneous algebraic reconstruction technique (ASART) in which a modified multilevel access scheme and an adaptive relaxation parameter adjustment method are developed to improve the quality of the reconstructed 3D structure. The reconstruction process is facilitated by using a column-sum substitution approach. This modified multilevel access scheme is adopted to arrange the order of projections so as to minimize the correlations between consecutive views within a limited angle range. In the adaptive relaxation parameter adjustment method, not only the weight matrix (as in the existing methods) but the gray levels of the pixels are employed to adjust the relaxation parameters so that the quality of the reconstruction is improved while the convergence process of the reconstruction is accelerated. In the column-sum substitution approach, the computation to obtain the reciprocal of the sum for the columns in each view is avoided so that the needed computations for each iteration can be reduced. Experimental results show that the proposed technique ASART is better based on objective quality measures than other methods, especially when data is noisy and limited in tilt angles. At the same time, the reconstruction by ASART outperforms that of simultaneous algebraic reconstruction technique (SART) in speed.

Determination of the volume-specific surface area by using transmission electron tomography for characterization and definition of nanomaterials

BackgroundTransmission electron microscopy (TEM) remains an important technique to investigate the size, shape and surface characteristics of particles at the nanometer scale. Resulting micrographs are two dimensional projections of objects and their interpretation can be difficult. Recently, electron tomography (ET) is increasingly used to reveal the morphology of nanomaterials (NM) in 3D. In this study, we examined the feasibility to visualize and measure silica and gold NM in suspension using conventional bright field electron tomography.ResultsThe general morphology of gold and silica NM was visualized in 3D by conventional TEM in bright field mode. In orthoslices of the examined NM the surface features of a NM could be seen and measured without interference of higher or lower lying structures inherent to conventional TEM. Segmentation by isosurface rendering allowed visualizing the 3D information of an electron tomographic reconstruction in greater detail than digital slicing. From the 3D reconstructions, the surface area and the volume of the examined NM could be estimated directly and the volume-specific surface area (VSSA) was calculated. The mean VSSA of all examined NM was significantly larger than the threshold of 60 m2/cm3.The high correlation between the measured values of area and volume gold nanoparticles with a known spherical morphology and the areas and volumes calculated from the equivalent circle diameter (ECD) of projected nanoparticles (NP) indicates that the values measured from electron tomographic reconstructions are valid for these gold particles.ConclusionThe characterization and definition of the examined gold and silica NM can benefit from application of conventional bright field electron tomography: the NM can be visualized in 3D, while surface features and the VSSA can be measured.

Fast and high-quality reconstruction in electron tomography based on an enhanced linear forward model

We study single-axis electron tomography and present an improved version of the linear forward model given by Fanelli and Öktem (2008 Inverse Problems 24 013001) which accounts for inelastic scattering and image distortions caused by imperfect optics. Based on the concept of approximate inverse, we derive an algorithm of filtered backprojection type which is much faster than the frequently used iterative methods. Numerical tests with simulated and measured transmission electron microscope data and comparisons with other FBP-type methods reveal that our algorithm provides reconstructions with high contrast and resolution, while the noise level is significantly reduced.

The Structure of a COPII Tubule

Nearly a third of all eukaryotic proteins are transported from the ER to the Golgi apparatus through the secretory pathway using COPII coated vesicles. Evidence suggests that this transport occurs via 500-900A vesicles that bud from the ER membrane. It has been shown that procollagen molecules utilize the COPII proteins for transport, but it is unclear how the COPII coat can accommodate these ∼3000A long molecules. We now present a cryogenic electron tomographic reconstruction of a Sec13/31 tubule that is approximately 3300A long containing a hollow cylindrical interior that is 300A in diameter, dimensions that are consistent with those that are required to encapsulate a procollagen molecule wrapped in a membrane and accessory COPII components. This structure suggests a novel mechanism that the COPII coat may employ to transport elongated cargo.

Exploring vitreous cryo-section-induced compression at the macromolecular level using electron cryo-tomography; 80S yeast ribosomes appear unaffected

Vitreous cryo-section-induced compression influences the interpretation and the reliability of electron microscopy images and tomographic reconstructions. Previous studies of this deformation have been focused at the cellular level where considerable compression occurs, yet the degree of possible intracellular macromolecular deformation has remained unclear. Here, electron cryo-tomographic reconstructions of vitreous cryo-sections show that 80S ribosomes, both intracellular and in an isolated state, appear able to resist section-induced compression. Our observations indicate that vitreous section-induced compression is non-uniform between whole cells that have been sectioned and their intracellular macromolecular complexes. We conclude that electron cryo-tomography of vitreous cryo-sections, in spite of section-induced compression, is a suitable technique for charting the structural organization of cellular nanomachines, such as ribosomes, in a cellular environment.

The structure of a COPII tubule

Nearly a third of all eukaryotic proteins are transported from the ER to the Golgi apparatus through the secretory pathway using COPII coated vesicles. Evidence suggests that this transport occurs via 500-900 Å vesicles that bud from the ER membrane. It has been shown that procollagen molecules utilize the COPII proteins for transport, but it is unclear how the COPII coat can accommodate these ∼3000 Å long molecules. We now present a cryogenic electron tomographic reconstruction of a Sec13/31 tubule that is approximately 3300 Å long containing a hollow cylindrical interior that is 300 Å in diameter, dimensions that are consistent with those that are required to encapsulate a procollagen molecule wrapped in a membrane and accessory COPII components. This structure suggests a novel mechanism that the COPII coat may employ to transport elongated cargo.

The platelet interior revisited: electron tomography reveals tubular α-granule subtypes

AbstractWe have used (cryo) electron tomography to provide a 3-dimensional (3D) map of the intracellular membrane organization of human platelets at high spatial resolution. Our study shows that the open canalicular system and dense tubular system are highly intertwined and form close associations in specialized membrane regions. 3D reconstructions of individual α-granules revealed large heterogeneity in their membrane organization. On the basis of their divergent morphology, we categorized α-granules into the following subtypes: spherical granules with electron-dense and electron-lucent zone containing 12-nm von Willebrand factor tubules, subtypes containing a multitude of luminal vesicles, 50-nm-wide tubular organelles, and a population with 18.4-nm crystalline cross-striations. Low-dose (cryo) electron tomography and 3D reconstruction of whole vitrified platelets confirmed the existence of long tubular granules with a remarkably curved architecture. Immunoelectron microscopy confirmed that these extended structures represent α-granule subtypes. Tubular α-granules represent approximately 16% of the total α-granule population and are detected in approximately half of the platelet population. They express membrane-bound proteins GLUT3 and αIIb-β3 integrin and contain abundant fibrinogen and albumin but low levels of β-thromboglobulin and no von Willebrand factor. Our 3D study demonstrates that, besides the existence of morphologically different α-granule subtypes, high spatial segregation of cargo exists within individual α-granules.