Bright-field scanning confocal electron microscopy using a double aberration-corrected transmission electron microscope

Scanning confocal electron microscopy (SCEM) offers a mechanism for three-dimensional imaging of materials, which makes use of the reduced depth of field in an aberration-corrected transmission electron microscope. The simplest configuration of SCEM is the bright-field mode. In this paper we present experimental data and simulations showing the form of bright-field SCEM images. We show that the depth dependence of the three-dimensional image can be explained in terms of two-dimensional images formed in the detector plane. For a crystalline sample, this so-called probe image is shown to be similar to a conventional diffraction pattern. Experimental results and simulations show how the diffracted probes in this image are elongated in thicker crystals and the use of this elongation to estimate sample thickness is explored.     

Three-dimensional imaging in double aberration-corrected scanning confocal electron microscopy, part I: elastic scattering

A transmission electron microscope fitted with both pre-specimen and post-specimen spherical aberration correctors enables the possibility of aberration-corrected scanning confocal electron microscopy. Imaging modes available in this configuration can make use of either elastically or inelastically scattered electrons. In this paper we consider image contrast for elastically scattered electrons. It is shown that there is no linear phase contrast in the confocal condition, leading to very low contrast for a single atom. Multislice simulations of a thicker crystalline sample show that sample vertical location and thickness can be accurately determined. However, buried impurity layers do not give strong, nor readily interpretable contrast. The accompanying paper examines the detection of inelastically scattered electrons in the confocal geometry.     

Three-dimensional imaging in double aberration-corrected scanning confocal electron microscopy, part II: inelastic scattering

The implementation of spherical aberration-corrected pre- and post-specimen lenses in the same instrument has facilitated the creation of sub-Angstrom electron probes and has made aberration-corrected scanning confocal electron microscopy (SCEM) possible. Further to the discussion of elastic SCEM imaging in our previous paper, we show that by performing a 3D raster scan through a crystalline sample using inelastic SCEM imaging it will be possible to determine the location of isolated impurity atoms embedded within a bulk matrix. In particular, the use of electron energy loss spectroscopy based on inner-shell ionization to uniquely identify these atoms is explored. Comparisons with scanning transmission electron microscopy (STEM) are made showing that SCEM will improve both the lateral and depth resolution relative to STEM. In particular, the expected poor resolution of STEM depth sectioning for extended objects is overcome in the SCEM geometry.     

Avoiding artefacts during electron microscopy of silver nanomaterials exposed to biological environments

Electron microscopy has been applied widely to study the interaction of nanomaterials with proteins, cells and tissues at nanometre scale. Biological material is most commonly embedded in thermoset resins to make it compatible with the high vacuum in the electron microscope. Room temperature sample preparation protocols developed over decades provide contrast by staining cell organelles, and aim to preserve the native cell structure. However, the effect of these complex protocols on the nanomaterials in the system is seldom considered. Any artefacts generated during sample preparation may ultimately interfere with the accurate prediction of the stability and reactivity of the nanomaterials. As a case study, we review steps in the room temperature preparation of cells exposed to silver nanomaterials (AgNMs) for transmission electron microscopy imaging and analysis. In particular, embedding and staining protocols, which can alter the physicochemical properties of AgNMs and introduce artefacts thereby leading to a misinterpretation of silver bioreactivity, are scrutinized. Recommendations are given for the application of cryogenic sample preparation protocols, which simultaneously fix both particles and diffusible ions. By being aware of the advantages and limitations of different sample preparation methods, compromises or selection of different correlative techniques can be made to draw more accurate conclusions about the data.     

Imaging friction tracks at diamond surfaces using reflection electron microscopy

Reflection electron microscopy (REM) is applied to image the structure of polished natural diamond (001) surfaces (of 5 × 4 mm size) after friction experiments under a pressure below the critical value. Friction tracks marked by a diamond needle after a single pass movement under a pressure of 13 GPa can be seen in REM images and show non-uniform contrast. The surface shows relatively dark image contrast at the light contacted area, which is possibly due to the structural modification at the top atomic layer. The high local contacting pressure pushes part of the needle into the surface which causes fracture, resulting in the formation of grooves at the surface. It is possible to have plastic deformation in this process, but no evidence has been found for the presence of cracking. The observations support the adhesion frictional mechanism rather than the micro-cleavage model.     

Theoretical aspects of image formation in the aberration-corrected electron microscope

The theoretical aspects of image formation in the transmission electron microscope (TEM) are outlined and revisited in detail by taking into account the elastic and inelastic scattering. In particular, the connection between the exit wave and the scattering amplitude is formulated for non-isoplanatic conditions. Different imaging modes are investigated by utilizing the scattering amplitude and employing the generalized optical theorem. A novel obstruction-free anamorphotic phase shifter is proposed which enables one to shift the phase of the scattered wave by an arbitrary amount over a large range of spatial frequencies. In the optimum case, the phase of the scattered wave and the introduced phase shift add up to −π/2 giving negative contrast. We obtain these optimum imaging conditions by employing an aberration-corrected electron microscope operating at voltages below the knock-on threshold for atom displacement and by shifting optimally the phase of the scattered electron wave. The optimum phase shift is achieved by adjusting appropriately the constant phase shift of the phase plate and the phase shift resulting from the defocus and the spherical aberration of the corrected objective lens. The realization of this imaging mode is the aim of the SALVE project (Sub-Å Low-Voltage Electron microscope).     

Reflection electron microscopy methods for the study of surface structure

The techniques of reflection electron microscopy (REM) using TEM instruments and scanning reflection electron microscopy (SREM) using STEM instruments have been explored as means for the observation of surface structure with high spatial resolution, better than 1 nm in each case. Under the ordinary environment of a commercial TEM instrument, we have studied the contrast in REM images of atomic steps and made comparison with the calculated results from the multi-slice dynamical diffraction theory. Comparison has also been made between the REM images of defects and the calculated images based on the column approximation. The influence of surface resonances on the contrast has been investigated. By SREM performed in a modified HB5 STEM with attached high vacuum preparation chamber, we have observed the formation of periodically distributed Pd particles on the surface of cleaved MgO.     

Review of the principal contrast effects observed at interphase boundaries using transmission electron microscopy

This review is concerned with the interpretation of contrast features observed at interphase interfaces using conventional transmission electron microscopy. The principal features which arise are interfacial dislocations and steps, four types of fringes, namely thickness, Δw, displacement and moiré fringes, and lattice images. The contrast mechanisms leading to these features are discussed in the framework of the dynamical theory of electron diffraction. In particular, the influence on image contrast of the cyrstal composition and structure, the interfacial configuration and the crystallographic relationship of the two phases in the specimen is considered. In general, electron diffraction in bi-crystalline specimens is complicated, and reliable interpretation can only be carried out when the diffraction conditions have been carefully established. The diffraction conditions most widely used are discussed, and their relative merits compared.     

Scanning electron microscopy comparison of the resin–dentin interface using different specimen preparation methods

Microscopy has been widely used to complement the data of studies related to dentin bonding; however, different specimen preparation methods may influence the analysis. Aiming to contribute to the reported scenario, this study evaluated the effect of two specimen‐sectioning methods (cleavage and diamond disk cut) on the quality of the scanning electron microscopy (SEM) images. Four crowns of human molars were selected and had an area of approximately 6 mm² of dentin exposed. They were then divided into two groups according to the universal adhesive application: total and self‐etching modes. Then, composite blocks were built up and the specimens were stored in deionized water to allow the postcuring. The specimens were further divided according to the sectioning method: cleavage or diamond disk cut. Four specimens were obtained from each tooth. They were desiccated, placed on aluminum stubs, sputter‐coated with gold, and observed in a scanning electron microscope, with ×2000 of magnification. The quality of the SEM images were evaluated by two calibrated examiners and classified into four scores (1–4). Mann–Whitney test (p < .05) showed that the diamond disk provided significantly higher scores than cleavage, whereas no significant difference was observed when comparing the total‐etching and self‐etching modes of application. The diamond disk cut method is preferable to the cleavage method to ensure the quality of the SEM analysis in studies involving the resin–dentin interface.     

Fundamental problems of imaging subsurface structures in the backscattered electron mode in scanning electron microscopy

In‐depth imaging of subsurface structures in scanning electron microscopy (SEM) is usually obtained by detecting backscattered electrons (BSE). For a layer‐by‐layer imaging in BSE microtomography, it is preferable to use an energy filtering of BSE. A simple approach is used to estimate the contrast by using backscattering coefficients of bulk materials and the maximum escape depths of the BSE. The contrast obtained by BSE energy filtering is about twice that of the standard BSE method by varying the acceleration voltage. The contrast decreases with increasing information depth. The information depth is about four times smaller than the electron range. The transmission of the spectrometer influences the minimum current of the order of 10^?8 A that is needed to get a contrast of 1%, for example.     

Scanning electron microscopy imaging of dislocations in bulk materials, using electron channeling contrast

The imaging and characterization of dislocations is commonly carried out by thin foil transmission electron microscopy (TEM) using diffraction contrast imaging. However, the thin foil approach is limited by difficult sample preparation, thin foil artifacts, relatively small viewable areas, and constraints on carrying out in situ studies. Electron channeling imaging of electron channeling contrast imaging (ECCI) offers an alternative approach for imaging crystalline defects, including dislocations. Because ECCI is carried out with field emission gun scanning electron microscope (FEG-SEM) using bulk specimens, many of the limitations of TEM thin foil analysis are overcome. This paper outlines the development of electron channeling patterns and channeling imaging to the current state of the art. The experimental parameters and set up necessary to carry out routine channeling imaging are reviewed. A number of examples that illustrate some of the advantages of ECCI over thin foil TEM are presented along with a discussion of some of the limitations on carrying out channeling contrast analysis of defect structures.     

Scanning electron microscopy of the capillary loops in the dermal papillae of the hand in primates,including man

The microvasculature of the skin of the hand in primates, including man, was examined by means of scanning electron microscopy of corrosion casts. In this study, the microvascular patterns and structures in different areas of the hand, and the changes in vascular patterns that occur with age, have been described. The typical structure of the capillary loops in the hand can be observed in the ball of the finger of the young adult monkey. The capillary loops were formed out of not just one capillary vessel, but two or three vessels. Each capillary vessel arose and divided into several branches at the papillae, and these became descending limbs. After the loop passed a hairpin turn, the descending limbs were 1.5 times larger than the ascending limbs in the intrapapillary portion, and they became extrapapillary venules. The descending limbs connected with the postcapillary venules in the postpapillary portion and with the horizontal network. The postcapillary venules fused with each other to form the primary and secondary venous arcades. The secondary venous arcades anastomosed with each other and flowed into the subpapillary venules, which run along the dermal furrow in the fingerprint. Changes in vascular patterns with age could be observed. In the infant fingerprint, the vascular systems had not yet differentiated, especially the venous system in the dermis. In the old adult finger, the capillary loops presented complicated features deviating due to aging.     

Reconstruction of the projected electrostatic potential in high-resolution transmission electron microscopy including phenomenological absorption

The projected electrostatic potential is reconstructed from a high-resolution exit wave function through a maximum-likelihood refinement algorithm. The theory of an already existing algorithm [1] is extended to include the effects of phenomenological absorption. Various tests with a simulated exit wave function of YBa₂Cu₃O₇ in [1 0 0] orientation used as a source show that the reconstruction is successful, regardless of the strongly differing scattering power of atomic columns, even for the case of strong dynamical diffraction. Object thickness, the amount of absorption, and a residual defocus aberration of the wave function—parameters often unknown or difficult to measure in experiments—can be determined accurately with the aid of the refinement algorithm in a self-consistent way. For the next generation of instruments, with information limits of 0.05 nm and better, reconstruction accuracies of better than 2% can be expected, which is sufficient to measure and display the structural and chemical information with the aid of an accurate projected potential map.     

High-resolution electron microscopy study on crystal structures of high-Tc superconductors

Recent studies of high-resolution electron microscopy on the high-T_c superconductors of Y-Ba-Cu-O and Bi-Ca-Sr-Cu-O are presented. The observed images of crystals thinner than 3 nm, taken under conditions that approached the Scherzer defocus condition, directly show the arrangements of cations and oxygen-vacant positions. The results reveal structural characteristics of the atomic scale; this offers important insights into the origin of the high-T_c superconductivity. The usefulness of high-resolution electron microscopy for studying complicated crystal structures is demonstrated for the high-T_c oxides.     

High-resolution electron microscopy and domain structures of pentagonal frank-kasper phases

The Laboratory of Atomic Imaging of Solids is dedicated to the direct imaging of materials in various fields of solid-state sciences at the atomic or molecular level. The main programs of this laboratory are briefly described, including quasicrystals, new phases and microdomains in Frank-Kasper phases, suboxides of metals, catalysts, minerals found in China, and molecular structure of organic substances. A recent systematic investigation of domain structures consisting of juxtaposed icosahedral columns is also presented.     

Preparation of neural tissues and sensory structures for electron microscopy

This paper describes the preparation of specimens of neural tissues and sensory structures for electron microscopy. A solution of 2% paraformaldehyde and 2.5% glutaraldehyde was used as fixative. The fixative entered the spinal cord of the rhesus monkey through the aorta abdominalis and emptied into the internal ear of the guinea pig via the arteria vertebralis by the vascular perfusion method. After perfusion, the spinal cord and internal ear were removed from the columna vertebralis and temporal bone, respectively. The tissues were postfixed in 1% OsO₄. If a desired direction of the section was required, directional embedding was applied. The application of vascular perfusion fixation, excising tissues on orientation, and embedding specimens in a certain direction makes it possible to obtain the desired tissue section and intact cellular ultrastucture.     

Single molecule microscopy methods for the study of DNA origami structures

Single molecule microscopy techniques play an important role in the investigation of advanced DNA structures such as those created by the DNA origami method. Three single molecule microscopy techniques are particularly interesting for the investigation of complex self-assembled three-dimensional (3D) DNA nanostructures, namely single molecule fluorescence microscopy, atomic force microscopy (AFM), and cryogenic transmission electron microscopy (cryo-EM). Here we discuss the strengths of these three techniques and demonstrate how their interplay can yield very important and unique new insights into the structure and conformation of advanced biological nanostructures. The applications of the three single molecule microscopy techniques are illustrated by focusing on a self-assembled DNA origami 3D box nanostructure. Its size and structure were studied by AFM and cryo-EM, while the lid opening, which can be controlled by the addition of oligonucleotide keys, was recorded by F?rster/fluorescence resonance energy transfer (FRET) spectroscopy.     

Probing rotation dynamics of biomolecules using polarization based fluorescence microscopy

Fluorescence polarization, particularly fluorescence anisotropy (FA) can be used to characterize the rotation dynamics and interactions of biomolecules. We report here fluorescence polarization microscopy based on a two-photon fluorescence microscope. Two-photon fluorescence excited by a linearly polarized fs laser beam was separated into components of parallel and perpendicular polarization and then recorded simultaneously by two detectors. From the images corresponding to different combinations of the polarization for the excitation and fluorescence photons, images of FA, or polarization difference, can be derived. It is demonstrated that FA microscopy is capable of probing rotational mobility of the fluorescent molecules and their interaction with the surroundings, but displays lower axial resolution than fluorescence intensity images. It is proved that the degraded axial resolution of FA imaging is intrinsic to the current experimental set-up. Artifacts in FA imaging of aligned molecules are also discussed.     

Simulations of scanning electron microscopy imaging and charging of insulating structures

We present a three‐dimensional simulation of scanning electron microscope (SEM) images and surface charging. First, the field above the sample is calculated using Laplace's equation with the proper boundary conditions; then, the simulation algorithm starts following the electron trajectory outside the sample by using electron ray tracing. When the electron collides with the specimen, the algorithm keeps track of the electron inside the sample by simulating the electron scattering history with a Monte Carlo code. During this phase, secondary and backscattered electrons are emitted to form an image and primary electrons are absorbed; therefore, a charge density is formed in the material. This charge density is used to recalculate the field above and inside the sample by solving the Poisson equation with the proper boundary conditions. Field equation, Monte Carlo scattering simulation, and electron ray tracing are therefore integrated in a self‐consistent fashion to form an algorithm capable of simulating charging and imaging of insulating structures. To maintain generality, this algorithm has been implemented in three dimensions. We shall apply the so‐defined simulation to calculate both the global surface voltage and local microfields induced by the scanning beam. Furthermore, we shall show how charging affects resolution and image formation in general and how its characteristics change when imaging parameters are changed. We shall address magnification, scanning strategy, and applied field. The results, compared with experiments, clearly indicate that charging and the proper boundary conditions must be included in order to simulate images of insulating features. Furthermore, we shall show that a three‐dimensional implementation is mandatory for understanding local field formation.