An Interactive Simulation and Visualization Tool for Conventional and Aberration-Corrected Transmission Electron Microscopy

BackgroundThe transmission electron microscope is used to acquire structural information of macromolecular complexes. However, as any other imaging device, it introduces optical aberrations that must be corrected if high-resolution structural information is to be obtained. The set of all aberrations are usually modeled in Fourier space by the so-called Contrast Transfer Function (CTF). Before correcting for the CTF, we must first estimate it from the electron micrographs. This is usually done by estimating a number of parameters specifying a theoretical model of the CTF. This estimation is performed by minimizing some error measure between the theoretical Power Spectrum Density (PSD) and the experimentally observed PSD. The high noise present in the micrographs, the possible local minima of the error function for estimating the CTF parameters, and the cross-talking between CTF parameters may cause errors in the estimated CTF parameters.ResultsIn this paper, we explore the effect of these estimation errors on the theoretical CTF. For the CTF model proposed in [1] we show which are the most sensitive CTF parameters as well as the most sensitive background parameters. Moreover, we provide a methodology to reveal the internal structure of the CTF model (which parameters influence in which parameters) and to estimate the accuracy of each model parameter. Finally, we explore the effect of the variability in the detection of the CTF for CTF phase and amplitude correction.ConclusionWe show that the estimation errors for the CTF detection methodology proposed in [1] does not show a significant deterioration of the CTF correction capabilities of subsequent algorithms. All together, the methodology described in this paper constitutes a powerful tool for the quantitative analysis of CTF models that can be applied to other models different from the one analyzed here.

Determining the extent of oxidation in batches of metal nanoparticles is essential to predict the behaviour of the material. Using aberration corrected transmission electron microscopy (TEM) it was possible to detect the formation of an oxide shell, of thickness 3 nm, on the surface of copper nanoparticles. Further analysis showed that this shell actually consists of two layers, both of which were polycrystalline in nature with domains in the size range of 1–2 nm, and having a thickness of 1.5 nm each. Energy dispersive x-ray spectroscopy confirms that the layers arise due to the formation of oxides, but it was not possible to determine their exact nature. Analysis of the intensity variation within images obtained via probe corrected scanning TEM combined with a high angle annular dark field detector indicates that the shell consists of an inner layer of cuprous oxide (Cu2O) and an outer layer of cupric oxide (CuO). This work was complemented by conventional TEM which provided size distribution and revealed that the majority of particles have a core consisting of a single crystal of copper. This demonstrates the ability of TEM to help to determine the oxidation state of nanoparticles and its potential to be applied to a wide range of homogenous and heterogeneous nanoparticles.

We have developed a spherical aberration corrected transmission electron microscopy(Cs-corrected TEM) technique that allows us to obtain clearer images in real space than everbefore. We applied this technique to titanium oxide, in which light elements such as oxygenare difficult to observe using TEM because of its small cross section and electronicdamage. In the present study, we successfully observed oxygen atoms in rutileTiO2. In addition, this direct observation of oxygen atoms enabled us to study the Magnéli structure(TinO2n−1), which is caused by oxygen vacancies. These vacancies caused an atomic relaxation of thetitanium and oxygen atoms. The relaxed atoms formed a characteristic shear structureof rutile titanium dioxide phase. This shear structure of the Magnéli structure(TinO2n−1) was visualized with a spatial resolution of 0.119 nm. At the same time, the selected areadiffraction (SAD) pattern of the defect structure was obtained. Additional spots wereshown inside the rutile [110] spot. We made structural models of the shear structureand simulated the diffraction pattern and images using a multi-slice simulation.Additional spots in the simulated diffraction patterns accurately reconstructed theexperimental data. We also considered the possibility of the real-space analysis of localstructures using spherical aberration corrected transmission electron microscopy.

Aberration measurement using the Ronchigram contrast transfer function

Image formation in phase-contrast electron microscopy is governed by the contrast transfer function (CTF). The key parameter to tune the CTF is the defocus. A precise and unbiased estimate of the defocus is essential to determine the forward model and interpret high resolution images in cryo-TEM. We present an algorithm based on the weak-phase approximation for determination of the defocus and astigmatism of the objective lens from recorded images of an amorphous sample. The algorithm identifies Thon rings in the power spectrum density (PSD) and uses them to estimate astigmatism and defocus together with their uncertainties. For the astigmatism estimation we use a transformation to polar coordinates to fit ellipses to the Thon rings. Averaging the PSD over these ellipses reduces CTF estimation to a 1D problem and enhances the signal-to-noise ratio.

IntroductionCurrently, the resolution of structural detail in conventional transmission electron microscopy (CTEM) of organic materials is limited by the electron beam damage suffered by the sample. It has recently been shown that the imaging modes now available in scanning transmission electron microscopy (STEM) provide the potential for improvements in resolution over CTEM methods. However, the effect of the sensitivity of an organic sample itself to beam damage has not been compared in STEM versus CTEM. In STEM each element of the sample area is briefly illuminated with a small diameter, high flux, scanning electron beam, whereas in CTEM the entire sample area is continuously illuminated with a large diameter, lower flux, static electron beam. Although it has been shown that the amount of sample damage in CTEM is independent of dose rate(2), the electron flux in STEM may range from 3 to 4 orders of magnitude greater than that in CTEM, with a possible influence on damage rate. Generally, the effects of beam size, high flux, and short dwell time of the scanning electron beam on sensitivity of the sample to damage in STEM are not known.

Interaction between single noble metal atoms and graphene edges has been investigated via aberration-corrected and monochromated transmission electron microscopy. A collective motion of the Au atom and the nearby carbon atoms is observed in transition between energy-favorable configurations. Most trapping and detrapping processes are assisted by the dangling carbon atoms, which are more susceptible to knock-on displacements by electron irradiation. Thermal energy is lower than the activation barriers in transition among different energy-favorable configurations, which suggests electron-beam irradiation can be an efficient way of engineering the graphene edge with metal atoms.

Structural and Electronic Properties of BaO/MgO(001)-type Interface Studied via Aberration-corrected Transmission Electron Microscopy and First-principles Calculations

We present a method that allows for a quantitative measurement of the surface self-diffusion on nanostructures, such as nanoparticles, at the atomic scale using aberration-corrected high-resolution transmission electron microscopy (HRTEM). The diffusion coefficient can be estimated by measuring the fluctuation of the atom column occupation at the surface of Au nanoparticles, which is directly observable in temporal sequences of HRTEM images. Both a Au icosahedron and a truncated Au octahedron are investigated, and their diffusion coefficients are found to be in the same order of magnitude, D = 10(-17) to 10(-16) cm(2)/s. It is to be assumed that the measured surface diffusion is affected by the imaging electron beam. This assumption is supported by the observed instability of a (5 × 1) surface reconstruction on a {100} Au facet.

We use conventional and aberration-corrected transmission electron microscopy (TEM) and ab initio calculations to investigate the structural and electronic properties of \ensuremath{\beta}-FeSi${}_{2}$ nanoparticles, which are a promising material for photovoltaic applications due to a band gap of 1 eV and a high absorption coefficient. The nanoparticles have average sizes of \ensuremath{\sim}20 nm, form aggregates, and are prepared by gas-phase synthesis. Amorphous SiO${}_{x}$ shells with thicknesses of \ensuremath{\sim}1.7 nm around \ensuremath{\beta}-FeSi${}_{2}$ cores are identified on individual nanoparticles using electron energy-loss spectroscopy, while stacking fault domains in the nanoparticles are observed using high-resolution TEM, nanobeam electron diffraction, and automated diffraction tomography. Ab initio calculations indicate only minor changes in band structure in the faulted structure when compared to perfect \ensuremath{\beta}-FeSi${}_{2}$. The optical properties of imperfect \ensuremath{\beta}-FeSi${}_{2}$ nanoparticles are therefore expected to be the same as those of the perfect structure, suggesting that \ensuremath{\beta}-FeSi${}_{2}$ nanoparticles are suitable candidates for use in optical absorber layers in thin film solar cells.

Since the invention of the transmission electron microscope (TEM) in 1931 by Max Knoll and Ernst Ruska, modern TEMs underwent remarkable developments in its overall stability and electron optics. In particular, correction of higher-order lens aberrations has facilitated structural characterization of single molecules at atomic resolution. This improvement in resolution has greatly assisted the design and development of new nano-objects in the field of materials and biological sciences. However, taking full advantage of these developments requires high chemical purity in the sample preparation technique and an electron transparent sample support that not only ensures the reliability in structural characterization, but also boosts the signal-to-noise ratio of TEM images. Moreover, the electron beam in TEMs, in addition to image formation, can be used for structural transformation due to the strong interaction of electrons with the investigated material. In this thesis, the structure of mass-selected molecular species, deposited on graphene via electro-spray ion beam deposition (ES-IBD), is investigated by aberration-corrected high-resolution transmission electron microscopy (AC-HRTEM) at acceleration voltages between 60 and 80 kV. Due to its impressive electronic and mechanical properties, single- layer graphene (SLG) is used as TEM substrate throughout this work. The transfer methodology of the SLG, obtained by mechanical exfoliation or chemical vapor deposition (CVD), to the TEM grids is presented, followed by thorough structural characterization to analyze the morphology and crystallinity of the transferred SLG using HRTEM. First, beam-sensitive organic molecules such as cytochrome-class hemeprotein (cytochrome-c), chaperonin complex protein molecule (GroEL), copper-pthalocyanine (CuPc) and com- plex molecular magnetic systems in the form of single molecule magnets (SMMs) were investigated. The results showed that despite the presence of the highly conductive substrate and the use of low electron acceleration voltages, small organic molecules did not survive. However, large globular proteins showed some level of electron resistance before suffering radiation damage. Next, the molecular structure of single phosphotungstic acid (PTA) anion molecules deposited by either ES-IBD or the drop-cast method was analyzed and compared. The results showed homogeneous morphology of the samples prepared by ES-IBD. Further- more, by varying coverage and landing energy, it was possible to modify the required morphology. This allowed better control of deposition as compared to the drop cast ap- proach. A main advantage of ES-IBD is the chemical purity during deposition. This is not guaranteed in the drop-cast technique which is performed under ambient conditions. The main part of this thesis deals with the electron-beam-induced transformation of alkali-iodide molecules (CsI, RbI, KI and NaI). The deposited alkali iodide nanoparticles were intentionally exposed to the electron beam to disintegrate them into single ions. It is found that during electron exposure these ions form regular clusters consisting of 4 to 7 atoms, or 2D-crystals. Notably, in the case of CsI, 2D-crystals form only on BLG, whereas on SLG only atomic clusters were formed. HRTEM in combination with electron energy-loss spectroscopy (EELS) is employed to identify the atomic species of the 2D crystals on the atomic scale. In the case of NaI, 2D-crystals form on SLG. Their crystal structure corresponds to the CsCl-type, which has not been reported before. The etching behavior of graphene in the presence of alkali-iodide species is observed in HRTEM experiments for all the deposited species (CsI, RbI, KI, and NaI). By mea- suring the increase in the perimeter of etched holes in SLG, the rate of etching has been calculated. We found that etching was faster in the presence of both alkali and halide ions, as compared to the presence of only one ionic species. First-principles DFT simulation studies were performed to understand the interaction of alkali-iodide dimers on SLG. These calculations yielded quantitative estimates on the charge transfer between the dimer species and the underlying SLG. It was found that adsorption of alkali-iodide on SLG reduces the bonding electron density between the carbon atoms, thus reducing the C-C binding energy. This effect makes the SLG susceptible to damage when exposed to 80-keV electrons, which establishes the catalytic behavior of alkali-iodide species as etchants of SLG.

Images taken by transmission electron microscopes are usually affected by lens aberrations and image defocus, among other factors. These distortions can be modeled in reciprocal space using the contrast transfer function (CTF). Accurate estimation and correction of the CTF is essential for restoring the high-resolution signal in cryogenic electron microscopy (cryoEM). Previously, we described the implementation of algorithms for this task in the cisTEM software package (Grant et al., 2018). Here we show that taking sample characteristics, such as thickness and tilt, into account can improve CTF estimation. This is particularly important when imaging cellular samples, where measurement of sample thickness and geometry derived from accurate modeling of the Thon ring pattern helps judging the quality of the sample. This improved CTF estimation has been implemented in CTFFIND5, a new version of the cisTEM program CTFFIND. We evaluated the accuracy of these estimates using images of tilted aquaporin crystals and eukaryotic cells thinned by focused ion beam milling. We estimate that with micrographs of sufficient quality CTFFIND5 can measure sample tilt with an accuracy of 3° and sample thickness with an accuracy of 5 nm.

The energy filtered imaging technique has so far been carried out in a scanning transmission electron microscope (STEM) fitted with a sector type energy analyzer. The STEM has a disadvantage of low beam parallelity because it uses a convergent beam, while the conventional transmission electron microscope (CTEM) allows good phase contrast and diffraction contrast to be obtained because of the high parallelity of the beam, and allows also high resolution images to be obtained. The technique to obtain energy filtered CTEM images has thus far been carried out by a Castaing-Henry type filter or an Ω type filter. However, these filters have the disadvantage of lower energy resolution than conventional sector type energy analyzer at the present time. This paper reports energy filtered CTEM images of MgO smoke, obtained using a new scanning CTEM image technique and a high energy resolution sector type energy analyzer which can resolve bulk and surface plasmon energy.

Multicolored gene reporters for light microscopy are indispensable for biomedical research, but equivalent genetic tools for electron microscopy (EM) are still rare despite the increasing importance of nanometer resolution for reverse engineering of molecular machinery and reliable mapping of cellular circuits. We here introduce the fully genetic encapsulin/cargo system of Quasibacillus thermotolerans (Qt), which in combination with the recently characterized encapsulin system from Myxococcus xanthus (Mx) enables multiplexed gene reporter imaging via conventional transmission electron microscopy (TEM) in mammalian cells. Cryo-electron reconstructions revealed that the Qt encapsulin shell self-assembles to nanospheres with T = 4 icosahedral symmetry and a diameter of ∼43 nm harboring two putative pore regions at the 5-fold and 3-fold axes. We also found that upon heterologous expression in mammalian cells, the native cargo is autotargeted to the inner surface of the shell and exhibits ferroxidase activity leading to efficient intraluminal iron biomineralization, which enhances cellular TEM contrast. We furthermore demonstrate that the two differently sized encapsulins of Qt and Mx do not intermix and can be robustly differentiated by conventional TEM via a deep learning classifier to enable automated multiplexed EM gene reporter imaging.

Correction of axial chromatic aberration improves the contrast transfer in bright-field phase contrast imaging