Reverse Monte Carlo reconstruction algorithm for discrete electron tomography based on HAADF‐STEM atom counting

In this paper, we propose an algorithm to obtain a three‐dimensional reconstruction of a single nanoparticle based on the method of atom counting. The location of atoms in three dimensions has been successfully performed using simulations of high‐angle‐annular‐dark‐field images from only three zone‐axis projections, [110], [310] and [211], for a face‐centred cubic particle. These three orientations are typically accessible by low‐tilt holders often used in high‐performance scanning transmission electron microscopes.     

Three‐dimensional analysis of intermediate filament networks using SEM tomography

We identified tomographic reconstruction of a scanning electron microscopy tilt series recording the secondary electron signal as a well‐suited method to generate high‐contrast three‐dimensional data of intermediate filament (IF) networks in pancreatic cancer cells. Although the tilt series does not strictly conform to the projection requirement of tomographic reconstruction, this approach is possible due to specific properties of the detergent‐extracted samples. We introduce an algorithm to extract the graph structure of the IF networks from the tomograms based on image analysis tools. This allows a high‐resolution analysis of network morphology, which is known to control the mechanical response of the cells to large‐scale deformations. Statistical analysis of the extracted network graphs is used to investigate principles of structural network organization which can be linked to the regulation of cell elasticity.     

A method for determining void arrangements in inverse opals

The periodic arrangement of voids in ceramic materials templated by colloidal crystal arrays (inverse opals) has been analysed by transmission electron microscopy. Individual particles consisting of an approximately spherical array of at least 100 voids were tilted through 90° along a single axis within the transmission electron microscope. The bright‐field images of these particles at high‐symmetry points, their diffractograms calculated by fast Fourier transforms, and the transmission electron microscope goniometer angles were compared with model face‐centred cubic, body‐centred cubic, hexagonal close‐packed, and simple cubic lattices in real and reciprocal space. The spatial periodicities were calculated for two‐dimensional projections. The systematic absences in these diffractograms differed from those found in diffraction patterns from three‐dimensional objects. The experimental data matched only the model face‐centred cubic lattice, so it was concluded that the packing of the voids (and, thus, the polymer spheres that composed the original colloidal crystals) was face‐centred cubic. In face‐centred cubic structures, the stacking‐fault displacementvector is . No stacking faults were observed when viewingthe inverse opal structure along the orthogonal <110>‐type directions, eliminating the possibility of a random hexagonally close‐packed structure for the particles observed. This technique complements synchrotron X‐ray scattering work on colloidal crystals by allowing both real‐space and reciprocal‐space analysis to be carried out on a smaller cross‐sectional area.     

Automated high-resolution three-dimensional fluorescence imaging of large biological specimens

We describe a novel automated technique for visualizing the three‐dimensional distribution of fluorochrome‐labelled components, in which image resolution is uncoupled from specimen size. This method is based on computer numerically controlled milling technology and combines an arrayed imaging technique with fluorescence capabilities. Fluorescent signals are segmented by emission spectra such that multiple fluorochromes present within a single specimen may be reconstructed and visualized individually or as a group. The automated nature of the system minimizes the workload and time involved in image capture and volume reconstruction. As an application, the system was used to image zones of fluorochrome‐labelled microdamage within an 8‐mm diameter cylinder of trabecular bone at a voxel size of 3 × 3 × 8 μm³. Our reconstruction of this specimen provides a visual map and quantitative measures of the volume of damage present throughout the cylinder, clearly demonstrating the interpretive power afforded by three‐dimensional visualization. The three‐dimensional nature of this highly automated and adaptable system has the potential to facilitate new diagnostic tools and techniques with application to a wide range of biological and medical research fields.     

Characterization of nitride thin films by electron backscatter diffraction

Thin films incorporating GaN, InGaN and AlGaN are presently arousing considerable excitement because of their suitability for UV and visible light‐emitting diodes and laser diodes. However, because of the lattice mismatch between presently used substrates and epitaxial nitride thin films, the films are of variable quality. In this paper we describe our preliminary studies of nitride thin films using electron backscattered diffraction (EBSD). We show that the EBSD technique may be used to reveal the relative orientation of an epitaxial thin film with respect to its substrate (a 90° rotation between a GaN epitaxial thin film and its sapphire substrate is observed) and to determine its tilt (a GaN thin film was found to be tilted by 13 ± 1° towards [101 0]_GaN), where the tilt is due to the inclination of the sapphire substrate (cut off‐axis by 10° from (0001)_sapphire towards (101 0)_sapphire). We compare EBSD patterns obtained from As‐doped GaN films grown by plasma‐assisted molecular beam epitaxy (PA‐MBE) with low and high As₄ flux, respectively. Higher As₄ flux results in sharper, better defined patterns, this observation is consistent with the improved surface morphology observed in AFM studies. Finally, we show that more detail can be discerned in EBSD patterns from GaN thin films when samples are cooled.     

3D reconstruction of histological sections: Application to mammary gland tissue

In this article, we present a novel method for the automatic 3D reconstruction of thick tissue blocks from 2D histological sections. The algorithm completes a high‐content (multiscale, multifeature) imaging system for simultaneous morphological and molecular analysis of thick tissue samples. This computer‐based system integrates image acquisition, annotation, registration, and three‐dimensional reconstruction. We present an experimental validation of this tool using both synthetic and real data. In particular, we present the 3D reconstruction of an entire mouse mammary gland and demonstrate the integration of high‐resolution molecular data. Microsc. Res. Tech. 73:1019–1029, 2010. © 2010 Wiley‐Liss, Inc.     

Automated high-throughput electron tomography by pre-calibration of image shifts

Electron tomography is a versatile method for obtaining three‐dimensional (3D) images with transmission electron microscopy. The technique is suitable to investigate cell organelles and tissue sections (100–500 nm thick) with 4–20 nm resolution. 3D reconstructions are obtained by processing a series of images acquired with the samples tilted over different angles. While tilting the sample, image shifts and defocus changes of several µm can occur. The current generation of automated acquisition software detects and corrects for these changes with a procedure that incorporates switching the electron optical magnification. We developed a novel method for data collection based on the measurement of shifts prior to data acquisition, which results in a five‐fold increase in speed, enabling the acquisition of 151 images in less than 20 min. The method will enhance the quality of a tilt series by minimizing the amount of required focus‐change compensation by aligning the optical axis to the tilt axis of the specimen stage. The alignment is achieved by invoking an amount of image shift as deduced from the mathematical model describing the effect of specimen tilt. As examples for application in biological and materials sciences 3D reconstructions of a mitochondrion and a zeolite crystal are presented.     

Method for 3D fibre reconstruction on a microrobotic platform

Automated handling of a natural fibrous object requires a method for acquiring the three‐dimensional geometry of the object, because its dimensions cannot be known beforehand. This paper presents a method for calculating the three‐dimensional reconstruction of a paper fibre on a microrobotic platform that contains two microscope cameras. The method is based on detecting curvature changes in the fibre centreline, and using them as the corresponding points between the different views of the images. We test the developed method with four fibre samples and compare the results with the references measured with an X‐ray microtomography device. We rotate the samples through 16 different orientations on the platform and calculate the three‐dimensional reconstruction to test the repeatability of the algorithm and its sensitivity to the orientation of the sample. We also test the noise sensitivity of the algorithm, and record the mismatch rate of the correspondences provided. We use the iterative closest point algorithm to align the measured three‐dimensional reconstructions with the references. The average point‐to‐point distances between the reconstructed fibre centrelines and the references are 20–30 μm, and the mismatch rate is low. Given the manipulation tolerance, this shows that the method is well suited to automated fibre grasping. This has also been demonstrated with actual grasping experiments.     

A convenient method for electron tomography sample preparation using a focused ion beam

Here we report a new sample preparation method for three‐dimensional electron tomography. The method uses the standard film deposition and focused ion beam (FIB) methods to significantly reduce the problems arising from the projected sample thickness at high tilt angles. The method can be used to prepare tomography samples that can be imaged up to a ±75° tilt range which is sufficient for many practical applications. The method can minimize the problem of Ga⁺ contamination, as compared to the case of FIB preparation of rod‐shaped samples, and provides extended thin regions for standard 2D projection analyses. Microsc. Res. Tech. 75:1165–1169, 2012. © 2012 Wiley Periodicals, Inc.     

Directional analysis of digitized three-dimensional images by configuration counts

A method for estimating the orientated rose of normal directions of a three‐dimensional (3D) set Z from a digitization of Z, i.e. a voxel image, is presented. It is based on counts of informative configurations in n×n×n voxel cubes. An algorithm for finding all informative configurations is proposed and an estimation procedure is described in detail for the case n= 2. The presented method is a 3D version of a method of estimating the orientated rose of binary planar images using n×n configurations. A new feature is the design‐based approach, being more appropriate for biomedical image analysis than the formerly applied model‐based approach.     

A method for structure analysis of nanomaterials by electron diffraction: Phase identification and unit cell determination

We report a quick and easy method for a random selected area electron diffraction (SAED) pattern without rotating and tilting the specimen to perform phase identification and unit cell determination by combined with the XRD softwares. If your TEM is well aligned and camera length is carefully corrected, two‐dimensional (2D)‐SAED pattern can be directly transformed to 1D‐profile after the center determination of pattern, this profile is then imported to XRD analysis packages. Finally, phase identification and unit cell determination can be performed after peak search or precise peak position determined by profile fitting. Two examples, flaky‐like TiO₂ nanomaterial and TiO₂ nanotubes precipitated by the silver nanoparticles, were tested and verified for the validation of phase identification and unit cell determination using this method; the successful crystallographic analysis of one single gold nanocrystal indicates it is still validate for the nanocrystals with the smaller diffraction volume, but need two or more random tilt SAED patterns. This method could be further used in the quantitative phase analysis, structure determination and Rietveld refinement for the nanomaterials if the reliable integrated intensity can be extracted. Microsc. Res. Tech. 76:641–647, 2013. © 2013 Wiley Periodicals, Inc.     

A software tool for tomographic axial superresolution in STED microscopy

A method for generating three‐dimensional tomograms from multiple three‐dimensional axial projections in STimulated Emission Depletion (STED) superresolution microscopy is introduced. Our STED< method, based on the use of a micromirror placed on top of a standard microscopic sample, is used to record a three‐dimensional projection at an oblique angle in relation to the main optical axis. Combining the STED< projection with the regular STED image into a single view by tomographic reconstruction, is shown to result in a tomogram with three‐to‐four‐fold improved apparent axial resolution. Registration of the different projections is based on the use of a mutual‐information histogram similarity metric. Fusion of the projections into a single view is based on Richardson‐Lucy iterative deconvolution algorithm, modified to work with multiple projections. Our tomographic reconstruction method is demonstrated to work with real biological STED superresolution images, including a data set with a limited signal‐to‐noise ratio (SNR); the reconstruction software (SuperTomo) and its source code will be released under BSD open‐source license.     

Evaluation of electron tomography reconstruction methods for interface roughness measurement

We evaluate the suitability of simultaneous iterative reconstruction technique (SIRT), filtered back projection, and simultaneous algebraic reconstruction technique methods for buried interface roughness measurements. We also investigate the effect of total electron dose distributed over the entire tilt series on measured roughness values. We investigate the applicability of the dose fractionation theorem by evaluating the effect of an increasing number of images, i.e., decreasing tilt increment size at fixed total electron irradiation dose on the quantitative measurement of buried interface roughness. The results indicate that SIRT is the most suitable method for reconstruction and a 3° to 5° angle is optimal for the roughness measurement.     

Three‐dimensional morphometric comparison of normal and apoptotic endothelial cells based on laser scanning confocal microscopy observation

Three‐dimensional (3D) morphometric analysis of cellular and subcellular structures provides an effective method for spatial cell biology. Here, 3D cellular and nuclear morphologies are reconstructed to quantify and compare morphometric differences between normal and apoptotic endothelial cells. Human umbilical vein endothelial cells (HUVECs) are treated with 60 μM H₂O₂ to get apoptotic cell model and then a series of sectional images are acquired from laser scanning confocal microscopy. The 3D cell model containing plasma membrane and cell nucleus is reconstructed and fused utilizing three sequential softwares or packages (Mimics, Geomagic, and VTK). The results reveal that H₂O₂ can induce apoptosis effectively by regulating the activity of apoptosis‐related biomolecules, including pro‐apoptotic factors p53 and Bax, and anti‐apoptotic factor Bcl‐2. Compared with the normal HUVECs, the apoptotic cells exhibit significant 3D morphometric parameters (height, volume and nucleus‐to‐cytoplasm ratio) variation. The present research provides a new perspective on comparative quantitative analysis associated with cell apoptosis and points to the value of LSCM as an objective tool for 3D cell reconstruction. Microsc. Res. Tech. 76:1154–1162, 2013. © 2013 Wiley Periodicals, Inc.     

An electron microscopy three‐dimensional characterization of titania nanotubes

To characterize complex, three‐dimensional nanostructures, modern microscopy techniques are needed, such as electron tomography and focused ion beam (FIB) sectioning. The aim of this study was to apply these two techniques to characterize TiO₂ nanotubes in terms of their size, shape, volume, porosity, geometric surface area, and specific surface area (SSA). For these experiments, titania nanotubes were fabricated by means of the electrochemical oxidation of titanium at a voltage of 20 V for 2 hr followed by heat treatment at 450°C for 3 hr to change the amorphous structure into a crystalline anatase structure. The quantitative data obtained from the FIB and electron tomography reconstructions show a high similarity in porosity and some differences in SSA. These might be the result of differences in resolution between the two reconstruction techniques.     

Depth enhancement of 3D microscopic living‐cell image using incoherent fluorescent digital holography

Multilayer images of living cells are typically obtained using confocal or multiphoton microscopy. However, limitations on the distance between consecutive scan layers hinder high‐resolution three‐dimensional reconstruction, and scattering strongly degrades images of living cell components. Consequently, when overlapping information from different layers is focused on a specific point in the camera, this causes uncertainty in the depiction of the cell components. We propose a method that combines the Fresnel incoherent correlation holography and a depth‐of‐focus reduction algorithm to enhance the depth information of three‐dimensional cell images. The proposed method eliminates overlap between light elements in the different layers inside living cells and limitations on the interlayer distance, and also enhances the contrast of the reconstructed holograms of living cells.     

Efficient three‐phase reconstruction of heterogeneous material from 2D cross‐sections via phase‐recovery algorithm

Digital reconstruction of a complex heterogeneous media from the limited statistical information, mostly provided by different imaging techniques, is the key to the successful computational analysis of this important class of materials. In this study, a novel approach is presented for three‐dimensional (3D) reconstruction of a three‐phase microstructure from its statistical information provided by two‐dimensional (2D) cross‐sections. In this three‐step method, first two‐point correlation functions (TPCFs) are extracted from the cross‐section(s) using a spectral method suitable for the three‐phase media. In the next step, 3D TPCFs are approximated for all vectors in a representative volume element (RVE). Finally, the 3D microstructure is realized from the full‐set TPCFs obtained in the previous step, using a modified phase‐recovery algorithm. The method is generally applicable to any complex three‐phase media, here illustrated for an SOFC anode microstructure. The capabilities and shortcomings of the method are then investigated by performing a qualitative comparison between example cross‐sections obtained computationally and their experimental equivalents. Finally, it is shown that the method almost conserves key microstructural properties of the media including tortuosity, percolation and three‐phase boundary length (TPBL).     

Microstructural analysis of iron aluminide formed by self-propagating high-temperature synthesis mechanism in aluminium matrix composite

An aluminium matrix composite with iron aluminide formed in situ as a result of self‐propagated high‐temperature synthesis was examined. The structural characteristics of the reinforcement investigated by scanning electron microscopy and transmission electron microscopy methods are presented. Iron aluminide particles with a very fine grain size and of two shapes, cubic and needle‐like, were observed. No differences in their phase composition were found by the selective electron diffraction pattern method. The composite reinforcement formed in the early stage of self‐propagating high‐temperature synthesis consisted only of the Al₃Fe phase.