Single-axis Tilt Research Articles

Tomographic subvolume alignment and subvolume classification applied to myosin V and SIV envelope spikes

Electron tomography is a technique for three-dimensional reconstruction, that is widely used for imaging macromolecules, macromolecular assemblies or whole cells. Combined with cryo-electron microscopy, it is capable of visualizing structural detail in a state close to in vivo conditions in the cell. In electron tomography, micrographs are taken while tilting the specimen to different angles about a fixed axis. Due to mechanical constraints, the angular tilt range is limited. As a consequence, the reconstruction of a 3D image is missing data, which for a single axis tilt series is called the "missing wedge", a region in reciprocal space where Fourier coefficients cannot be obtained experimentally. Tomographic data is analyzed by extracting subvolumes from the raw tomograms, by alignment of the extracted subvolumes, multivariate data analysis, classification, and class-averaging, which results in an increased signal-to-noise ratio and substantial data reduction. Subvolume analysis is a valuable tool to discriminate heterogeneous populations of macromolecules, or conformations of a macromolecule or macromolecular assembly as well as to characterize interactions between macromolecules. However, this analysis is hampered by the lack of data in the original tomograms caused by the missing wedge. Here, we report enhancements of our subvolume processing protocols in which the problem of the missing data in reciprocal space is addressed by using constrained correlation and weighted averaging in reciprocal space. These procedures are applied to the analysis of myosin V and simian immunodeficiency virus (SIV) envelope spikes. We also investigate the effect of the missing wedge on image classification and establish limits of reliability by model calculations with generated phantoms.

Three-dimensional architecture of murine rod outer segments determined by cryoelectron tomography

The rod outer segment (ROS) of photoreceptor cells houses all components necessary for phototransduction, a set of biochemical reactions that amplify and propagate a light signal. Theoretical approaches to quantify this process require precise information about the physical boundaries of the ROS. Dimensions of internal structures within the ROS of mammalian species have yet to be determined with the precision required for quantitative considerations. Cryoelectron tomography was utilized to obtain reliable three-dimensional morphological information about this important structure from murine retina. Vitrification of samples permitted imaging of the ROS in a minimally perturbed manner and the preservation of substructures. Tomograms revealed the characteristic highly organized arrangement of disc membranes stacked on top of one another with a surrounding plasma membrane. Distances among the various membrane components of the ROS were measured to define the space available for phototransduction to occur. Reconstruction of segments of the ROS from single-axis tilt series images provided a glimpse into the three-dimensional architecture of this highly differentiated neuron. The reconstructions revealed spacers that likely maintain the proper distance between adjacent discs and between discs and the plasma membrane. Spacers were found distributed throughout the discs, including regions that are distant from the rim region of discs.

Multiple‐axis tomography: applications to basal bodies from Paramecium tetraurelia

Transmission electron tomography is becoming a powerful tool for studying subcellular components of cells. Classical approaches for electron tomography consist of recording images along a single-tilt axis. This approach is being improved by dual-axis reconstructions and/or high-tilt devices (tilt angle>+/-60 degrees) on microscopes to compensate part of the information loss due to the 'missing wedge' phenomena. In the present work we have evaluated the extension of the dual-axis technique to a multiple-axis approach, and we demonstrate a freely available plug-in for the Java-based freeware image-analysis software ImageJ. Our results from phantom and experimental data sets from Paramecium tetraurelia epon-embedded sections have shown that multiple-axis tomography achieves results equivalent to those obtained by dual-axis approach without the requirement for high-tilt devices. This new approach allows performance of high-resolution tomography, avoiding the need for high-tilt devices, and therefore will increase the access of electron tomography to a larger community.

Electron tomography of microelectronic device interconnects

Abstract As the dimensions of microelectronic devices continue to decrease, single transmission electron microscopy images are not able to properly represent the 3D structures when the structure's curvature is comparable to the sample thickness. Electron tomography was used to study cylindrical vias coated with Ta-barrier layers and Cu-seed layers in 3D. Tomography reconstructions from both bright field images and high angle annular dark field images are presented. Fidelity of the reconstruction from single-axis and dual-axis tilt series is compared. Strategies for improving the fidelity of the reconstruction and making electron tomography practically applicable for device failure analysis of microelectronic industry are discussed.

Novel Analytical Method of Nanoparticle Dispersibility in Polymer Nanocomposites; TEM-CT and 3D Topological Analysis

We established a quantitative analytical method of nanoparticle dispersibility, which includes the following steps; 3D reconstructed image by TEM-CT, particle separation by 3D image analysis, and calculation of a dispersibility index. First the 3D image was reconstructed by a series of 121 projections collected along a single tilt axis on TEM. Second particles were separated from the reconstructed image by binarization. The distance between the gravity centers of particles was measured from the reconstructed images. Finally a dispersibility index was defined as the average of the shortest distances for adjacent nanoparticles divided by an ideal distance. The proposed method was applied for alumina nanoparticles in polymer nanocomposites under different synthetic processes, and the dispersibility of them was calculated by the index. The correlation between the dispersibility as a structure and the transmittance at 700 nm as a function was found to be linear. This method is applicable to evaluate the 3D dispersibility quantitatively for the nanoparticle arrays.

Electron Tomography of Microelectronic Device Interconnects

Abstract As the dimensions of microelectronic devices continue to decrease, single transmission electron microscopy images are not able to properly represent the 3D structures when the structure's curvature is comparable to the sample thickness. Electron tomography was used to study cylindrical vias coated with Ta-barrier layers and Cu-seed layers in 3D. Tomography reconstructions from both bright field images and high angle annular dark field images are presented. Fidelity of the reconstruction from single-axis and dual-axis tilt series is compared. Strategies for improving the fidelity of the reconstruction and making electron tomography practically applicable for device failure analysis of microelectronic industry are discussed.

Optimal Determination of Particle Orientation, Absolute Hand, and Contrast Loss in Single-particle Electron Cryomicroscopy

A computational procedure is described for assigning the absolute hand of the structure of a protein or assembly determined by single-particle electron microscopy. The procedure requires a pair of micrographs of the same particle field recorded at two tilt angles of a single tilt-axis specimen holder together with the three-dimensional map whose hand is being determined. For orientations determined from particles on one micrograph using the map, the agreement (average phase residual) between particle images on the second micrograph and map projections is determined for all possible choices of tilt angle and axis. Whether the agreement is better at the known tilt angle and axis of the microscope or its inverse indicates whether the map is of correct or incorrect hand. An increased discrimination of correct from incorrect hand (free hand difference), as well as accurate identification of the known values for the tilt angle and axis, can be used as targets for rapidly optimizing the search or refinement procedures used to determine particle orientations. Optimized refinement reduces the tendency for the model to match noise in a single image, thus improving the accuracy of the orientation determination and therefore the quality of the resulting map. The hand determination and refinement optimization procedure is applied to image pairs of the dihydrolipoyl acetyltransferase (E2) catalytic core of the pyruvate dehydrogenase complex from Bacillus stearothermophilus taken by low-dose electron cryomicroscopy. Structure factor amplitudes of a three-dimensional map of the E2 catalytic core obtained by averaging untilted images of 3667 icosahedral particles are compared to a scattering reference using a Guinier plot. A noise-dependent structure factor weight is derived and used in conjunction with a temperature factor ( B=−1000 Å 2) to restore high-resolution contrast without amplifying noise and to visualize molecular features to 8.7 Å resolution, according to a new objective criterion for resolution assessment proposed here.

MEMS-based 14 GHz resolution dynamic optical filter

A 64-channel 14 GHz-spaced MEMS-based filter with flat-top passbands and 7 dB insertion loss has been constructed. It utilises a lens and grating to demultiplex the light onto an array of electrostatically actuated micromechanical single axis tilt mirrors.

Electron Spectroscopic Tomography for Materials Science

Abstract Tomographic 3D-reconstructions from tilt series of projections differ in two major aspects when moving from biochemistry (e.g.) to materials science applications: (I) Crystalline inorganic materials fail the weak phase image formation approximation and suffer from dynamic and nonlinear contrast contributions (which depend for both CTEM and HREM on atomic lattice plane orientation). (II) exposure tolerance increases by two orders of magnitude. We therefore propose energy filtered tomography with inelastic electrons (electron spectroscopic imaging, ESI) as a new method for 3D-reconstruction of free or buried subsurface nanostructures or nanoparticles. Energy filters are not used here to improve SNR by removing inelastic background, but to provide the necessary projection relationship between a 3D concentration distribution and the (processed) image intensity. Ideally, a 4-dimensional data space is recorded for a single tilt axis tomography experiment, D=D(δE, Θ,x,y), (with 5E as energy loss, Θ as tilt, and (x,y) as image coordinates). The material has to withstand exposures of T = t × Ntilt × Nloss (t = 5...20sec per image, Ntilt >12 directions, Nloss = 5....20 energy loss values).

Three-Dimensional Structure of Neurospora Mitochondria: New Insights Provided by Electron Tomography of the Frozen-Hydrated Organelles

Abstract Electron tomography is the most widely applicable method for obtaining three-dimensional information by electron microscopy. It is, in fact, the only method suitable for investigating pleomorphic structures, such as many supramolecular assemblies, organelles and cells. With the recent development of automated low-dose data-acquisition schemes, it is now possible to study molecules and cells embedded in vitreous ice. This opens up new horizons for investigating the functional organization of cellular components with minimal perturbation of the cellular context. In the present study we used automated electron tomography in conjunction with cryomicroscopy to reveal the internal organization and ultrastructure of mitochondria. The whole isolated cell organelles from Neurospora crassa were quick-frozen and examined in vitrified ice. Single-axis tilt series were recorded with a Philips CM 120 Biotwin under low-dose conditions; the estimated total exposure was 10,000 e−nm−2. The tilt increment was 1° and the tilt series ranged from −65° to +65°. In order to enhance the contrast of the rather thick samples zero-loss energy filtering was employed. The images were recorded with a Ik × Ik CCD camera. The experimental setup has been described by Koster et al2 Three-dimensional reconstructions were performed by weighted backprojection. Prior to the three-dimensional visualization of 3-D data sets a denoising technique based on nonlinear anisotropic diffusion was applied (Figs. 2, 3).

Three-Dimensional Reconstruction from Reduced Sets of Very Noisy Images Acquired Following a Single-Axis Tilt Schema: Application of a New Three-Dimensional Reconstruction Algorithm and Objective Comparison with Weighted Backprojection

In this work we propose a reconstruction algorithm (ART with blobs) that has not been previously used in electron Tomography and we compare it with the standard method in the field (weighted back projection, WBP). We assume that only a limited set of very noisy images, collected around a single axis tilt, is available; which is a typical situation in Electron Tomography. In general, the reconstruction problem is underdetermined (due to the limited number of projections) and the data are inconsistent (due to the high level of noise). The evaluation of the results is performed in a rigorous way by a task-oriented approach which makes use of numerical observers. ART with blobs outperforms WBP for a number of key tasks. Results are presented both for simplified line integral data and for realistic simulations of macromolecular structures embedded in amorphous ice.

Use of High-Performance Computing Algorithms in Combination With Parallel Computing for Tomography

The electron microscope is essential for resolving complex biological structures, such as mitochondria, which are too small to be viewed in detail with the light microscope. In contrast to a conventional instrument, the High-Voltage Electron Microscope (HVEM) located at the National Center for Microscopy and Imaging Research (NCMIR) can obtain images from relatively thick specimens that contain substantial three-dimensional structure. Though a single image acquired with the HVEM represents a projection through the specimen, tomographic methods can be applied to a set of images acquired from different orientations to derive a three-dimensional representation of its biological structure. Tomography requires extensive computation and considerable processing time on conventional workstations in order to reconstruct the typically large HVEM volumes from the tilt series.In order to expedite tomographic processing, we have implemented both the commonly used singleaxis tilt, R-weighted backprojection algorithm and two iterative reconstruction methods, algebraic reconstruction (ART) and simultaneous iterative reconstruction (SIRT) on the massively parallel Intel Paragon at the San Diego Supercompter Center.

Electron Tomography of Neuronal Mitochondria: Three-Dimensional Structure and Organization of Cristae and Membrane Contacts

The structure of neuronal mitochondria from chick and rat was examined using electron microscope tomography of chemically fixed tissue embedded in plastic and sliced in ≈500-nm-thick sections. Three-dimensional reconstructions of representative mitochondria were made from single-axis tilt series acquired with an intermediate voltage electron microscope (400 kV). The tilt increment was either 1° or 2° ranging from −60° to +60°. The mitochondrial ultrastructure was similar across species and neuronal regions. The outer and inner membranes were each ≈7 nm thick. The inner boundary membrane was found to lie close to the outer membrane, with a total thickness across both membranes of ≈22 nm. We discovered that the inner membrane invaginates to form cristae only through narrow, tubular openings, which we call crista junctions. Sometimes the cristae remain tubular throughout their length, but often multiple tubular cristae merge to form lamellar compartments. Punctate regions, ≈14 nm in diameter, were observed in which the inner and outer membranes appeared in contact (total thickness of both membranes ≈14 nm). These contact sites are known to a play a key role in the transport of proteins into the mitochondrion. It has been hypothesized that contact sites may be proximal to crista junctions to facilitate transport of proteins destined for the cristae. However, our statistical analyses indicated that contact sites are randomly located with respect to these junctions. In addition, a close association was observed between endoplasmic reticulum membranes and the outer mitochondrial membrane, consistent with the reported mechanism of transport of certain lipids into the mitochondrion.

Tomography without Tilt: Three-Dimensional Imaging of Microtubule/Motor Complexes

Usually structures such as microtubules are supposed to have surface lattices built from families of continuous helices, giving electron micrographs that can be analyzed by helical diffraction theory with a view to obtaining three-dimensional reconstructions. In the case of microtubules the helical surface lattice may be interupted by discontinuities, called seams, in which case the usual helical reconstruction approach is no longer applicable. Even so, by virtue of their “superhelical” protofilaments, microtubules are still helical structures and we use this feature to treat a microtubule image as a set of projections equivalent to images obtained in a single axis tilt series. The main thrust of this article is to discuss how to obtain images and image parameters best suited to a tomographic approach to three-dimensional reconstruction. The method is tested by comparing helical and back-projection reconstructions of appropriate microtubules both with and without surface lattice decoration by kinesin family motor proteins. Tomographic reconstruction gives an independent demonstration thatin vitroassembled microtubules have a B-type surface lattice. We show that 3-start, 15-protofilament microtubules have a seam, whereas 4-start microtubules have no seam and possess complete helical symmetry. Monomer motor domains attach to the outer ridge of the protofilaments and extend along the protofilament toward the plus end.

Energy filtered electron tomography of ice-embedded actin and vesicles

Semiautomatic single-axis tilt electron tomography has been used to visualize the three-dimensional organization of actin filaments in "phantom cells," i.e. lipid vesicles. The instrumentation consisted of a 120-kV electron microscope equipped with a postcolumn energy filter, which was used in the zero-loss imaging mode. Apart from changing the tilt angle, all steps required for automated tomography, such as recentering the image area, refocusing, and centering the energy-selecting slit, were performed by external computer control. This setup permitted imaging of ice-embedded samples up to a thickness of 800 nm with improved image contrast compared with that produced by tomography with a conventional electron microscope. In spite of the missing-wedge effect that is especially obvious in the study of membrane-filament interaction, single-axis tilt tomography was found to be an appropriate (in fact the only available) method for this kind of investigation. In contrast to random actin networks found in actin gels, actin filaments in and on vesicles with a bending radius of less than approximately 2 microns tend to be arranged in single layers of parallel filaments and often induce an elongated shape of the vesicles. Actin filaments located on the outside usually associate with the vesicle membrane.

Electron microscopic tomography: A tool for probing the structure and function of subcellular components

Tomography is a powerful tool for obtaining three-dirotation geometry. Although single-axis data collection is stanmensional information from transmission electron microscopy, but its dard in biomedical imaging (e.g., CAT scan, scanning via compuapplication faces unique challenges. A single-axis tilt geometry for terized axial tomography), other data collection geometries are data collection results in anisotropic resolution because full angular also routinely used. coverage is not feasible for most specimen preparations. This effect In the last 10 years, electron tomography has proved to be an can be minimized by combining two single-axis tilt series that have effective tool for 3D structure–function determination in cellular been collected orthogonal to each other. Rapid freezing has been and molecular biology (reviewed in [6–8]) . Recent successful successfully used to preserve the native structure of biological speciapplications include determining the location of microtubule initimens in a form that can be visualized in the high-vacuum environment ation sites in centrosomes isolated from fruit fly embryos [9] , required for electron microscopy; however, these preparations are extremely labile to electron exposure. As a result, application of todemonstrating dynamic changes that occur in muscle Z bands mography to frozen-hydrated specimens has only recently become with contraction [10], characterizing the arrangement and morfeasible with the development of automatic data collection, and with phology of crystals formed during the initial events of bone fora renewed appreciation for the principle of dose fractionation. Even mation [11,12], and providing new insight into the folding of with the limitations of traditional specimen preparations and convenDNA into chromosomes [13,14]. Here we briefly review the tional methods of data collection, electron microscopic tomography methodology, including recent efforts to overcome radiation damhas been successfully employed to probe the structure and function age and an unfavorable specimen geometry, and present a new of several important cellular components. Current efforts include comapplication where electron tomography is combined with videobining electron microscopic tomography and video-enhanced light enhanced light microscopy to correlate structural determination microscopy to correlate ultrastructural variation with the direction of with motion. chromosome motion during mitosis. q 1997 John Wiley & Sons, Inc. Int J Imaging Syst Technol, 8, 175–187, 1997

Tomography of paracrystalline specimens

3-D image reconstruction of objects with no symmetry are produced by combining images of specimens tilted at finely spaced intervals about a single tilt axis. Image fiducials, usually gold particles applied to the surface of the specimen, are used to orient and align the separate views and 3-D images calculated by back projection methods. Electron microscope tomography has developed into a powerful method for producing a 3-D image of objects lacking crystalline symmetry. Crystalline objects are usually reconstructed using Fourier based methods that produce averaged 3-D images. Electron crystallography has become a powerful tool for solving the 3-D structure of proteins at atomic resolution.Paracrystalline objects lie between crystalline and nonperiodic specimens because part of their structure is often well ordered but they have as well a significant disordered part. Muscle is one of the more notable members of this type of specimen because often the actin and myosin filaments form a well ordered array but the crossbridges, i.e. the motor domain of myosin, are much more disordered.

Three Dimensional imaging of biological macromolecules

3D imaging of both cellular structures as well as molecular assemblies of biological molecules has become an increasingly useful tool to study structure-function relationships of biological systems. In this paper instrumental and methodological developments are discussed towards automated 3D imaging, which will be illustrated by examples of structures studied in Martinsried. To image individual structures with dimensions in the range of 10-500 nm with a resolution of 1-5 nm, electron tomography is the only technique available. The strategy of choice depends on size and shape of the structure to be reconstructed. Single-tilt axis tomography is suitable for the reconstruction of unique structures (for example, irregularly shaped viruses or cellular structures). Random conical-tilt data collection, as well as angular reconstitution techniques, can be used to reconstruct the average structure of many copies of a particle, such as those present in suspension of one kind of protein. To reconstruct a unique structure with single-tilt axis tomography the tilt range and tilt increments are chosen to meet the resolution desired within the constraint of the allowable electron doses (Table 1).

A marker-free alignment method for electron tomography

In electron tomography of biological specimens, fiducial markers are normally used to achieve accurate alignment of the input projections. We address the problem of alignment of projections from objects that are freely supported and do not permit the use of markers. To this end we present a new alignment algorithm for single-axis tilt geometry based on the principle of Fourier-space common lines. An iterative scheme has been developed to overcome the noise-sensitivity of the common-line method. This algorithm was used to align a data set that was not amenable to alignment with fiducial markers.

Stereoscopy by tilted illumination in transmission electron microscopy

We describe a new technique for stereoscopic observation in transmission electron microscopy, employing tilted illumination. A triple-hole objective aperture is used so that the bright beam can pass through with or without tilt. Stereo views can be acquired by tilting the illumination such that the bright beam passes through a pair of symmetrically arranged apertures alternately. The advantages of this technique as compared to the commonly used method of single-axis tilt are: (i) greater speed, potentially at or close to video rate so that live 3D observation is possible; (ii) elimination of specimen movement associated with stage tilt; and (iii) perspective views corresponding to multiple tilting axes which can be realized by installing more aperture-holes and choosing their positions properly. The main limitation is that the angle of tilt is limited by the extent to which the astigmatism, introduced by beam tilt, can be compensated by the objective lens stigmators.