Distribution of projection angles for single-axis-tilt electron microscope tomography of extended thin planar specimens

An optimized distribution of tilt angles for tomography of specimens of non-circular cross-section is derived and tested with reconstructions of a phantom model.     

Quantitative comparison of energy-filtering transmission electron microscopy and atom probe tomography

Whereas transmission electron microscopy (TEM) is a well established method for the analysis of thin film structures down to the sub-nanometer scale, atom probe tomography (APT) is less known in the microscopy community. In the present work, local chemical analysis of sputtered Fe/Cr multilayer structures was performed with energy-filtering transmission electron microscopy (EFTEM) and APT. The single-layer thickness was varied from 1 to 6 nm in order to quantify spatial resolution and chemical sensitivity. While both the methods are able to resolve the layer structure, even at 2 nm thickness, it is demonstrated that the spatial resolution of the APT is about a factor of two, higher in comparison with the unprocessed EFTEM data. By calculating the influence of the instrumental parameters on EFTEM images of model structures, remaining interface roughness is indicated to be the most important factor that limits the practical resolution of analytical TEM.     

Towards automated electron holographic tomography for 3D mapping of electrostatic potentials

Electron-holographic tomography (EHT), that is, the combination of off-axis electron holography with electron tomography, was successfully applied for the quantitative 3D mapping of electrostatic potentials at the nanoscale. Here we present the first software package (THOMAS) for semi-automated acquisition of holographic tilt series, a prerequisite for efficient data collection. Using THOMAS, the acquisition time for a holographic tilt series, consisting of object and reference holograms, is reduced by a factor of five on average, compared to the previous, completely manual approaches. Moreover, the existing software packages for retrieving amplitude and phase information from electron holograms have been extended, now including a one-step procedure for holographic tilt series reconstruction. Furthermore, a modified SIRT algorithm (WSIRT) was implemented for the quantitative 3D reconstruction of the electrostatic potential from the aligned phase tilt series. Finally, the application of EHT to a polystyrene latex sphere test-specimen and a pn-doped Ge ‘needle’-shaped specimen are presented, illustrating the quantitative character of EHT. For both specimens the mean inner potential (MIP) values were accurately determined from the reconstructed 3D potential. For the Ge specimen, additionally the ‘built-in’ voltage across the pn junction of 0.5 V was obtained.     

Morphology of organic electronic materials imaged via electron tomography

Organic electronic materials and nanostructures have been studied with the use of electron tomography. Nanostructured materials including contrast enhancing features have been studied and double tilt data collection has been employed to improve reconstructions. Tomography reconstructions of active layers of organic solar cells, where various preparation techniques have been used, have been analysed and compared to device behaviour. Small changes in preparation procedures may lead to large differences in morphology and device performance, and the results also indicate a complex relation between these.     

Nanoscale scanning transmission electron tomography

Electron tomography enables the study of complex three‐dimensional objects with nanometre resolution. In materials science, scanning transmission electron microscopy provides images with minimal coherent diffraction effects and with high atomic number contrast that makes them ideal for electron tomographic reconstruction. In this study, we reviewed the topic of scanning transmission electron microscopy‐based tomography and illustrated the power of the technique with a number of examples with critical dimensions at the nanoscale.     

Surface rendering in electron tomography: A heuristic approach

The final result of an electron tomographic reconstruction, computed from several projections, is a 3-D array of data which resides in the computer memory as a long string (from 32³ to 256³) of numbers. It is vital to retrieve information relevant to biology from this vast amount of numbers, and to share it with the scientific community. This is done with the use of visualization processes that may represent the most time consuming task in a reconstruction. The present article describes a technique of surface rendering designed by the authors to represent structures characterized by the presence of a number of subunits and of features such as branching, holes and cavities. The software used produces a ‘structure’ (an array containing different types of variables, peculiar to the C programming language) that is able to exploit the resources of the graphic engines embedded in modern workstations and of graphic libraries. This structure can be used to produce visual presentations in the form of wireframe- and surface-rendered models with shading determined by the Gouraud algorithm.     

Limitations of beam damage in electron spectroscopic tomography of embedded cells

Elemental mapping in the energy filtering transmission electron microscope (EFTEM) can be extended into three dimensions (3D) by acquiring a series of two‐dimensional (2D) core‐edge images from a specimen oriented over a range of tilt angles, and then reconstructing the volume using tomographic methods. EFTEM has been applied to imaging the distribution of biological molecules in 2D, e.g. nucleic acid and protein, in sections of plastic‐embedded cells, but no systematic study has been undertaken to assess the extent to which beam damage limits the available information in 3D. To address this question, 2D elemental maps of phosphorus and nitrogen were acquired from unstained sections of plastic‐embedded isolated mouse thymocytes. The variation in elemental composition, residual specimen mass and changes in the specimen morphology were measured as a function of electron dose. Whereas 40% of the total specimen mass was lost at doses above 10⁶ e^?/nm², no significant loss of phosphorus or nitrogen was observed for doses as high as 10⁸ e^?/nm². The oxygen content decreased from 25 ± 2 to 9 ± 2 atomic percent at an electron dose of 10⁴ e^?/nm², which accounted for a major component of the total mass loss. The specimen thickness decreased by 50% after a dose of 10⁸ e^?/nm², and a lateral shrinkage of 9.5 ± 2.0% occurred from 2 × 10⁴ to 10⁸ e^?/nm². At doses above 10⁷ e^?/nm², damage could be observed in the bright field as well in the core edge images, which is attributed to further loss of oxygen and carbon atoms. Despite these artefacts, electron tomograms obtained from high‐pressure frozen and freeze‐substituted sections of C. elegans showed that it is feasible to obtain useful 3D phosphorus and nitrogen maps, and thus to reveal quantitative information about the subcellular distributions of nucleic acids and proteins.     

Ultrathin formvar support films for transmission electron microscopy

We have found that ultrathin Formvar films are easily and reliably made at an air-water interface by the drop method. By varying the concentration of Formvar in the drop, films of different characteristics can be obtained. Concentrations of 0.25–0.4% in ethylene dichloride produce extremely flat, ultrathin, and stable films that are especially suited for shadowed and negatively stained preparations. Low concentrations (? 0.1%) produce nets consisting of many tiny holes which, after carbon stabilization, are ideal for supporting high-resolution samples. Above 0.5%, films made by the drop method develop bubbles, and this bubble defect makes them unsuitable for section support. For section support, Formvar films made by the stripping method off mica are far superior to those made off glass. The films are more uniform in surface contour and thickness. They are less readily attacked by alcohols. Consequently, they are more resistant to staining procedures involving organic solvents and continue to be strong and uniform for section support.     

3-D reconstruction of the atomic positions in a simulated gold nanocrystal based on discrete tomography: prospects of atomic resolution electron tomography

A novel reconstruction procedure is proposed to achieve atomic resolution in electron tomography. The method exploits the fact that crystals are discrete assemblies of atoms (atomicity). This constraint enables us to obtain a three-dimensional (3-D) reconstruction of test structures from less than 10 projections even in the presence of noise and defects. Phase contrast transmission electron microscopy (TEM) images of a gold nanocrystal were simulated in six different zone axes. The discrete number of atoms in every column is determined by application of the channelling theory to reconstructed electron exit waves. The procedure is experimentally validated by experiments with gold samples. Our results show that discrete tomography recovers the shape of the particle as well as the position of its 309 atoms from only three projections.

Experiments on a nanocrystal that contains several missing atoms, both on the surface and in the core of the nanocrystal, while considering a high noise level in each simulated image were performed to prove the stability of the approach to reconstruct defects. The algorithm is well capable of handling structural defects in a highly noisy environment, even if this causes atom count “errors” in the projection data.     

Controlled microaspiration for high-pressure freezing: a new method for ultrastructural preservation of fragile and sparse tissues for TEM and electron tomography

High‐pressure freezing is the preferred method to prepare thick biological specimens for ultrastructural studies. However, the advantages obtained by this method often prove unattainable for samples that are difficult to handle during the freezing and substitution protocols. Delicate and sparse samples are difficult to manipulate and maintain intact throughout the sequence of freezing, infiltration, embedding and final orientation for sectioning and subsequent transmission electron microscopy. An established approach to surmount these difficulties is the use of cellulose microdialysis tubing to transport the sample. With an inner diameter of 200 μm, the tubing protects small and fragile samples within the thickness constraints of high‐pressure freezing, and the tube ends can be sealed to avoid loss of sample. Importantly, the transparency of the tubing allows optical study of the specimen at different steps in the process. Here, we describe the use of a micromanipulator and microinjection apparatus to handle and position delicate specimens within the tubing. We report two biologically significant examples that benefit from this approach, 3D cultures of mammary epithelial cells and cochlear outer hair cells. We illustrate the potential for correlative light and electron microscopy as well as electron tomography.     

Automated electron tomography with scanning transmission electron microscopy

We report the successful implementation of a fully automated tomographic data collection system in scanning transmission electron microscopy (STEM) mode. Autotracking is carried out by combining mechanical and electronic corrections for specimen movement. Autofocusing is based on contrast difference of a focus series of a small sample area. The focus gradient that exists in normal images due to specimen tilt is effectively removed by using dynamic focusing. An advantage of STEM tomography with dynamic focusing over TEM tomography is its ability to reconstruct large objects with a potentially higher resolution.     

Comparison of single‐particle analysis and electron tomography approaches: an overview

Three‐dimensional structure of a wide range of biological specimens can be computed from images collected by transmission electron microscopy. This information integrated with structural data obtained with other techniques (e.g., X‐ray crystallography) helps structural biologists to understand the function of macromolecular complexes and organelles within cells. In this paper, we compare two three‐dimensional transmission electron microscopy techniques that are becoming more and more related (at the image acquisition level as well as the image processing one): electron tomography and single‐particle analysis. The first one is currently used to elucidate the three‐dimensional structure of cellular components or smaller entire cells, whereas the second one has been traditionally applied to structural studies of macromolecules and macromolecular complexes. Also, we discuss possibilities for their integration with other structural biology techniques for an integrative study of living matter from proteins to whole cells.     

Comparing electron tomography and HRTEM slicing methods as tools to measure the thickness of nanoparticles

Nanoparticles’ morphology is a key parameter in the understanding of their thermodynamical, optical, magnetic and catalytic properties. In general, nanoparticles, observed in transmission electron microscopy (TEM), are viewed in projection so that the determination of their thickness (along the projection direction) with respect to their projected lateral size is highly questionable. To date, the widely used methods to measure nanoparticles thickness in a transmission electron microscope are to use cross-section images or focal series in high-resolution transmission electron microscopy imaging (HRTEM “slicing”). In this paper, we compare the focal series method with the electron tomography method to show that both techniques yield similar particle thickness in a range of size from 1 to 5 nm, but the electron tomography method provides better statistics since more particles can be analyzed at one time. For this purpose, we have compared, on the same samples, the nanoparticles thickness measurements obtained from focal series with the ones determined from cross-section profiles of tomograms (tomogram slicing) perpendicular to the plane of the substrate supporting the nanoparticles. The methodology is finally applied to the comparison of CoPt nanoparticles annealed ex situ at two different temperatures to illustrate the accuracy of the techniques in detecting small particle thickness changes.     

Optimization of STEM tomography acquisition — A comparison of convergent beam and parallel beam STEM tomography

In this paper two imaging modes in a state-of-the-art scanning transmission electron microscope (STEM) are compared: conventional STEM with a convergent beam (referred to as nanoprobe) and STEM with a parallel beam (referred to as microprobe). The effect and influence of both modes with respect to their depth of field are investigated. Tomograms of a human white blood cell (hemophagocytes) are acquired, aligned, and evaluated. It is shown that STEM using a parallel beam produces tomograms with fewer distortions and artifacts that allows resolving finer features. Microprobe STEM tomography is advantageous especially in life science, when semi-thin sections (approximately 0.5 μm thick) of biological samples are imaged at relatively low magnification with a large field of view.     

Focused ion beam scanning electron microscopy in biology

Since the end of the last millennium, the focused ion beam scanning electron microscopy (FIB‐SEM) has progressively found use in biological research. This instrument is a scanning electron microscope (SEM) with an attached gallium ion column and the 2 beams, electrons and ions (FIB) are focused on one coincident point. The main application is the acquisition of three‐dimensional data, FIB‐SEM tomography. With the ion beam, some nanometres of the surface are removed and the remaining block‐face is imaged with the electron beam in a repetitive manner. The instrument can also be used to cut open biological structures to get access to internal structures or to prepare thin lamella for imaging by (cryo‐) transmission electron microscopy. Here, we will present an overview of the development of FIB‐SEM and discuss a few points about sample preparation and imaging.     

TEM, HRTEM, electron holography and electron tomography studies of γ' and γ" nanoparticles in Inconel 718 superalloy

The aim of the study was the identification of γ' and γ" strengthening precipitates in a commercial nickel-base superalloy Inconel 718 (Ni-19Fe-18Cr-5Nb-3Mo-1Ti-0.5Al-0.04C, wt %) using TEM dark-field, HRTEM, electron holography and electron tomography imaging. To identify γ' and γ" nanoparticles unambiguously, a systematic analysis of experimental and theoretical diffraction patterns were performed. Using HRTEM method it was possible to analyse small areas of precipitates appearance. Electron holography and electron tomography techniques show new possibilities of visualization of γ' and γ" nanoparticles. The analysis by means of different complementary TEM methods showed that γ" particles exhibit a shape of thin plates, while γ' phase precipitates are almost spherical.     

A fast surface reconstruction method for fluorescence molecular tomography based on cross-beam edge back projection

An accurate surface reconstruction method is important to fluorescence molecular tomography (FMT) for it provides boundary information of the domain occupied by the image object which is essential to modeling light propagation in free space and inside the object. In this paper, a method based on cross-beam edge back projection (CEBP) is proposed to achieve fast and three-dimensional (3D) surface reconstruction for FMT. This method consists of a cost effective and easy-to-implement setup; it back-projects the edge of an image object of all projection angles along the actual light propagation path to perform 3D surface reconstruction. Simulation studies and experiments were performed to compare the reconstruction accuracy and computational cost of the CEBP based method and the conventional radon transform (RT) based method. Results demonstrate that the CEBP based method significantly accelerates surface reconstruction compared with the RT based method while keeping similar accuracy.     

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.