Dual-axis Tilt Series Research Articles

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.

AuTom-dualx: a toolkit for fully automatic fiducial marker-based alignment of dual-axis tilt series with simultaneous reconstruction.

MotivationDual-axis electron tomography is an important 3 D macro-molecular structure reconstruction technology, which can reduce artifacts and suppress the effect of missing wedge. However, the fully automatic data process for dual-axis electron tomography still remains a challenge due to three difficulties: (i) how to track the mass of fiducial markers automatically; (ii) how to integrate the information from the two different tilt series; and (iii) how to cope with the inconsistency between the two different tilt series.ResultsHere we develop a toolkit for fully automatic alignment of dual-axis electron tomography, with a simultaneous reconstruction procedure. The proposed toolkit and its workflow carries out the following solutions: (i) fully automatic detection and tracking of fiducial markers under large-field datasets; (ii) automatic combination of two different tilt series and global calibration of projection parameters; and (iii) inconsistency correction based on distortion correction parameters and the consequently simultaneous reconstruction. With all of these features, the presented toolkit can achieve accurate alignment and reconstruction simultaneously and conveniently under a single global coordinate system.Availability and implementationThe toolkit AuTom-dualx (alignment module dualxmauto and reconstruction module ) are accessible for general application at http://ear.ict.ac.cn, and the key source code is freely available under request.Supplementary information Supplementary data are available at Bioinformatics online.

The ASTRA Toolbox: A platform for advanced algorithm development in electron tomography

We present the ASTRA Toolbox as an open platform for 3D image reconstruction in tomography. Most of the software tools that are currently used in electron tomography offer limited flexibility with respect to the geometrical parameters of the acquisition model and the algorithms used for reconstruction. The ASTRA Toolbox provides an extensive set of fast and flexible building blocks that can be used to develop advanced reconstruction algorithms, effectively removing these limitations. We demonstrate this flexibility, the resulting reconstruction quality, and the computational efficiency of this toolbox by a series of experiments, based on experimental dual-axis tilt series.

Scanning Transmission Electron Tomography of Blood Platelets in Thick Sections

Electron tomography in the scanning transmission electron microscope (STEM) can be performed on sections of stained plastic-embedded tissues or cells of 1 to 2 micrometer thickness without effects of chromatic aberration because there are no imaging lenses after the specimen. By using a small STEM probe convergence angle of 1-2 mrad the geometrical broadening of the probe is restricted, which enables a spatial resolution of a few nanometers. Furthermore, by using an axial bright-field detector instead of the standard high-angle annular dark-field detector, image blurring due to multiple elastic scattering can be reduced in the lower part of the specimen. Here, we have applied STEM tomography to elucidate the 3D ultrastructure of human blood platelets, which are small anucleate blood cells that aggregate to seal leaks at sites of vascular injury and are important in the pathology of atherosclerosis and other diseases. Of particular interest are the morphological changes that occur in alpha-granules, which contain important proteins released when platelets are activated. Axial bright-field STEM electron tomographic tilt series were acquired at an accelerating voltage of 300 kV from 1.5-micrometer thick sections of platelets that had been prepared by rapid freezing and freeze-substitution; and the tomograms were reconstructed from dual-axis tilt series. The tomographic reconstructions revealed changes in ultrastructure that occurred on platelet activation including release of alpha granules through channels connecting to the plasma membrane. The research was supported by the intramural program of the National Institute of Biomedical Imaging and Bioengineering, and the research in the Storrie laboratory was supported in part by NIH grant R01 HL119393.

3D Tomographic Segmentation of Adult Cardiac Ventricle reveals a Complex Tubular and Vesicular Network surrounding the Gap Junction Plaque. Ultrastructure of the Connexome

<h3>Background</h3> The cardiac intercalated disc (ID) has been extensively studied by conventional transmission electron microscopy (EM). Yet, novel methods for tissue preservation (high-pressure freezing), image (3D tomographic EM) and analysis (image segmentation) that greatly improve image quality/resolution, have not been applied to the ID. Recent studies show that, at the ID, the gap junction protein Connexin43 is part of an interactome (a "connexome"). Here, we provide a structural characterization of the connexome. <h3>Methods</h3> Adult mouse ventricular tissue was prepared by high-pressure freezing and freeze substitution and embedded in resin. 200 nm thick sections were imaged with a 200kV electron microscope (FEI TF20). Images were collected at a set magnification of 9.6k on a 4kx4k CCD camera set to 2x binning, giving an effective pixel size of 1.76 nm. Dual-axis tilt series (1º steps, ±70º per axis) were acquired using SerialEM. Protomo software was used for aligning projection images and reconstructing tomograms. Visualization/segmentation of objects of interest was performed in Amira. <h3>Results</h3> In addition to classic ID structures, we observed (a) close proximity between gap junctions and mitochondria of opposing cells; (b) a complex network of tubular structures running perpendicular to the long cell axis; these structures showed a hollow interior, with an estimated inner diameter of ~40 nm and were often adjacent to gap junctions or desmosomes; (c) triads formed by lateral edges of gap junctions and desmosomes, with a rough budding vesicle separating the two structures; (d) budding vesicles of approximately 50 nm interrupting the continuity of one side of the gap junction plaque; (e) vesicular bodies of approx. 65 nm in diameter in the intercellular space, and in proximity to gap junction-containing regions. <h3>Conclusions</h3> We describe the nanometric landscape that surrounds gap junctions. We speculate that the connexome includes a physical association with molecules of the mitochondria, desmosome and microtubular network, and propose that microsomes may pass from one cell to another at the ID. Functional characterization of these structures may lead to novel clues as to the mechanisms of inherited or acquired arrhythmias that involve disruption of the ID.

Marker-Free Alignment of Dual-Axis Tilt Series and Subvolume Analysis of Data from Dual-Axis Tomograms

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

Marker-free dual-axis tilt series alignment

Dual-axis tilt series in electron tomography are collected by successively tilting the object about two approximately orthogonal tilt axes. Here we report on the extension of marker-free image registration based on cross-correlation techniques to dual-axis tilt series. A simultaneous geometry refinement yields accurate parameters for the computation of the final reconstruction. Both, image registration and 3D-reconstruction operate on the combined data from the paired single axis series rather than computing individual single axis tomograms followed by a separate combination step. We show that with simultaneous registration and reconstruction of dual-axis tilt series, tomograms with higher resolution are obtained than by merging separately computed tomograms.

Three‐Dimensional Analysis and Computer Modeling of the Capillary Endothelial Vesicular System with Electron Tomography

We examined the three-dimensional organization of the endothelial vesicular system with TEM tomography of semi-thick sections. Mouse abdominal muscle capillaries were perfused with terbium to label vesicular compartments open to the luminal surface. The tissue was prepared for TEM and semi-thick (250 nm) sections were cut. Dual axis tilt series, collected from +60° to -60° at 1° increments, were acquired in regions of labeled abluminal caveolae. These tomograms were reconstructed and analyzed to reveal three-dimensional vesicular associations not evident in thin sections. Reconstructed tomograms revealed free vesicles, both labeled and unlabeled, in the endothelial cytoplasm as well as transendothelial channels that spanned the luminal and abluminal membranes. A large membranous compartment connecting the luminal and abluminal surfaces was also present. Computer modeling of tomographic data and video animations provided three-dimensional perspectives to these structures. Uncertainties associated with other three-dimensional methods to study the capillary wall are remedied by tomographic analysis of semi-thick sections. Transendothelial channels of fused vesicles and free cytoplasmic vesicles give credence to their role as large pores in the transport of solutes across the walls of continuous capillaries.

High-angle triple-axis specimen holder for three-dimensional diffraction contrast imaging in transmission electron microscopy

Electron tomography requires a wide angular range of specimen-tilt for a reliable three-dimensional (3D) reconstruction. Although specimen holders are commercially available for tomography, they have several limitations, including tilting capability in only one or two axes at most, e.g. tilt-rotate. For amorphous specimens, the image contrast depends on mass and thickness only and the single-tilt holder is adequate for most tomographic image acquisitions. On the other hand, for crystalline materials where image contrast is strongly dependent on diffraction conditions, current commercially available tomography holders are inadequate, because they lack tilt capability in all three orthogonal axes needed to maintain a constant diffraction condition over the whole tilt range. We have developed a high-angle triple-axis (HATA) tomography specimen holder capable of high-angle tilting for the primary horizontal axis with tilting capability in the other (orthogonal) horizontal and vertical axes. This allows the user to trim the specimen tilt to obtain the desired diffraction condition over the whole tilt range of the tomography series. To demonstrate its capabilities, we have used this triple-axis tomography holder with a dual-axis tilt series (the specimen was rotated by 90° ex-situ between series) to obtain tomographic reconstructions of dislocation arrangements in plastically deformed austenitic steel foils.

Simultaneous alignment of dual-axis tilt series

We present a strategy for the alignment of dual-axis tomographic series, based on reference points and simultaneous alignment of both series. Each series is first aligned individually, an affine transformation is determined to bring the two series in a unique reference system, and all experimental coordinates are combined in a single system of equations. In case of severe shrinkage, a global and a local refinement of the orientation parameters are performed to correct all minors misalignments. The strategy is illustrated on tomographic experiments performed on sections from plastic-embedded biological samples. The efficiency in correcting the misalignment of gold particles and in improving the quality of the reconstruction is documented both visually and quantitatively. In our approach every region of the tomogram is associated with its own orientation parameters and can be eventually reconstructed with the preferred algorithm. This is convenient in the computation of 3D averages of equivalent structures. A simulation experiment is presented to show that the performances of this approach are superior to those of the method of rotation in direct space.

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.

A “flip–flop” rotation stage for routine dual-axis electron cryotomography

Electron cryotomography can be used to solve the three-dimensional structures of individual large macromolecules, assemblies, and even small intact cells to medium (∼4–8 nm) resolution in a near-native state, but restrictions in the range of accessible views are a major limitation. Here we report on the design, characterization, and demonstration of a new “flip–flop” rotation stage that allows facile and routine collection of two orthogonal tilt-series of cryosamples. Single- and dual-axis tomograms of a variety of samples are compared to illustrate qualitatively the improvement produced by inclusion of the second tilt-series. Exact quantitative expressions are derived for the volume of the remaining “missing pyramid” in reciprocal space. When orthogonal tilt-series are recorded to ±65° in each direction, as this new cryostage permits, only 11% of reciprocal space is left unmeasured. The tomograms suggest that further improvement could be realized, however, through better software to align and merge dual-axis tilt-series of cryosamples.

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.