Nanobeam electron diffraction and high resolution imaging analysis of InN films grown on sapphire

A JEOL JEM-3000F field emission, analytical, high-resolution transmission electron microscope (HRTEM) was used to study InN films grown on sapphire substrates. It was found that, while the InN films maintained the hexagonal (wurtzite) structure, InN nanodomains with a cubic (zincblende) structure were also formed in the films. Nanobeam electron diffraction techniques were applied for identification of the cubic phase. The identification of the cubic InN was confirmed by HRTEM structural imaging. The cubic InN nanodomains are 3-10 nm in diameter, and are orientated in two different orientations with their [110](cubic) and [110](cubic) axes parallel to each other and their (111)(cubic) planes parallel to the (0001)(hex) plane of the hexagonal InN.     

Instrumental requirements for high resolution imaging

The resolution of modern transmission electron microscopes reaches the physical limits imposed by lens aberrations and energy width. One of the many conditions to be fulfilled, the alignment of illuminating and imaging beam onto the coma-free objective axis, is particularly discussed here since axial coma cannot be detected by the usual resolution-checking methods. Space consumption of specimen stages prevents the full utilization of the magnetic saturation limit only in the 100 keV range. With higher energies, this handicap is obviated, and some additional advantages can be gained which promote material investigations at atomic resolution, and which are presently utilized in instrumental research projects. High resolution with biological specimens has up to now been unsuccessful because of radiation damage. Optimum utilization of all electrons scattered at the specimen must thus be given priority over optical resolution. Important instrumental requirements are minimum exposure beam control, imaging modes with high collection efficiency, and recording devices with high detection quantum efficiency connected on-line to image processors. A remarkable decrease in beam sensitivity of organic crystals, by more than one order, has been found by cooling the specimen down to 4 K which, by the use of superconducting lenses, can be combined with both ultra high vacuum and the stability requirements for high resolution. Yet up to now, such protection has not been achieved with He cryostates in conventional lenses, perhaps because a temperature increase even of only a few degree K is harmful. Purely magnetic imaging energy filters are about to be developed to a high optical quality but have been employed so far in only a few high resolution instruments. Such filters allow removal of the inelastic background and thus improvement of contrast of images of low-Z specimens, particularly in the dark field mode. Finally, some ‘non-conventional’ projects have made progress. Correction of spherical and chromatic aberration by multipole lenses offers a chance to improve remarkably the resolution in the 100 keV range, to extend the bandwidth of phase contrast transfer and to obtain highly resolved information about inelastic images when an energy filter is also applied. Electron holography provides possibly useful large area phase contrast, particularly if the electron energy is decreased, which may be of great benefit in investigations of unstained specimens.     

Image matching between experimental and simulated high‐resolution electron micrographs of sapphire on the orientation

The effects of imaging parameters have been studied on their roles of the severe mismatches between experimental and simulated high‐resolution transmission electron micrographs of sapphire along the direction. Image simulation and convergent‐beam electron diffraction techniques have been performed on misalignments of the electron beam and the crystal specimen. Based on this study, we have introduced an approach to achieve reliable simulation for experimental images of sapphire on the projection by the use of iterative digital image matching.     

Mapping of ELNES on a nanometre scale by electron spectroscopic imaging

The amorphous interfacial layer between Si substrates and diamond films grown by plasma-assisted chemical vapour deposition has been studied by electron spectroscopic imaging. The amorphous layer consists mainly of carbon, which can only be distinguished from the diamond film by analysis of the near-edge structure (ELNES) of the carbon K edge. Series of electron spectroscopic images were acquired across the carbon K edge and were analysed in order to reveal the presence of the π*- and σ*-excitations. After background removal from the corresponding images, phase maps for the distribution of sp² and sp³ hybridized carbon can be obtained. From the whole series of images, electron energy-loss spectra can be extracted for any given area in the images. The results show that the amorphous layer covers large areas along the interface and that regions with only 1–2 nm layer thickness can clearly be analysed. The results obtained with the electron spectroscopic imaging technique will be compared with results obtained on a field emission gun scanning transmission electron microscope.     

Quantitative high resolution transmission electron microscopy of nanostructured semiconductors

Peak‐finding procedures and the geometric phase method of quantitative high resolution electron microscopy (qHRTEM) were applied to determine the local strain and the chemical composition of nanostructured semiconductor materials. The growth of the structures investigated was induced by minimization of strain energy. The analysis of strain distribution is necessary for the understanding of the self‐organized formation of nanostructures. The possibilities and limitations of the methods are discussed in detail by analysing HRTEM images of (Si,Ge) islands and of a double layer of stacked quantum dots of (In,Ga)As and Ga(Sb,As).     

Atomic imaging using secondary electrons in a scanning transmission electron microscope: experimental observations and possible mechanisms

We report detailed investigation of high-resolution imaging using secondary electrons (SE) with a sub-nanometer probe in an aberration-corrected transmission electron microscope, Hitachi HD2700C. This instrument also allows us to acquire the corresponding annular dark-field (ADF) images both simultaneously and separately. We demonstrate that atomic SE imaging is achievable for a wide range of elements, from uranium to carbon. Using the ADF images as a reference, we studied the SE image intensity and contrast as functions of applied bias, atomic number, crystal tilt, and thickness to shed light on the origin of the unexpected ultrahigh resolution in SE imaging. We have also demonstrated that the SE signal is sensitive to the terminating species at a crystal surface. A possible mechanism for atomic-scale SE imaging is proposed. The ability to image both the surface and bulk of a sample at atomic-scale is unprecedented, and can have important applications in the field of electron microscopy and materials characterization.     

Transmission electron microscopy method providing high resolution in the cell section without radiation damage

Several new features of mitochondrial nucleoid and its surroundings in mammalian cells were described previously (Pracha?, 2016). Very small details were observed using the improved transmission electron microscopy method, as described in the article. In the meantime, the method has again been improved to 2 Å resolutions in the cell section. The method described in detail in the present work is documented on the same records that were published in lower resolution in the work Pracha? (2016), enabling comparison of the achieved resolution with the previous one. New records are also presented, showing extremely high resolution and thus implying the importance of the method. Potential use of this method in different fields is suggested.     

Characterization of ultrafine coal fly ash particles by energy-filtered TEM

In this study, energy-filtered transmission electron microscopy is demonstrated to be a valuable tool for characterizing ultrafine coal fly ash particles, especially those particles encapsulated in or associated with carbon. By examining a series of elemental maps (K-edge maps of C and O, and L-edge maps of Si, Al, Ti and Fe) recorded using the three-window method, considerable numbers of titanium and iron species with sizes from several nanometres to submicrometre were shown to be present, typically as oxides dispersed in the carbonaceous matrix. Crystalline phases, such as rutile and iron-rich oxide spinel, were also identified from electron diffraction patterns and high-resolution TEM images. Information about these ultrafine coal fly ash particles regarding their size, morphology, elemental composition and distribution, and crystalline phases, which has not been available previously in conventional ash studies, should be useful in toxicological studies and related environmental fields.     

Towards sub-A atomic resolution electron diffraction imaging of metallic nanoclusters: a simulation study of experimental parameters and reconstruction algorithms

A simulation study is carried out to elucidate the effects of dynamical scattering, electron beam convergence angle and detection noise on atomic resolution diffraction imaging of small particles and to develop effective reconstruction procedures. Au nanoclusters are used as model because of their strong scattering. The results show that the dynamical effects of electron diffraction place a limit on the size of Au nanoclusters that can be reconstructed from the diffraction intensities with sufficient accuracy. For smaller Au nanoclusters, the simulations show that diffraction patterns recorded under the experimental conditions can be reconstructed using a combination of phase retrieval algorithms. The use of a low-resolution image is shown to be effective for reconstructing diffraction patterns without the central beam. A new algorithm for estimating the object support is proposed.     

Polyphosphate at the Streptomyces lividans cytoplasmic membrane is enhanced in the presence of the potassium channel KcsA

The distribution of polyphosphate (polyP) within the cytoplasmic membrane of Streptomyces lividans hyphae or protoplasts has been determined at high spatial resolution by elemental mapping using energy‐filtered electron microscopy (EFTEM). The results revealed that polyP was best traceable after its interaction with lead ions followed by their precipitation as lead sulphide. Concomitant studies of the S.lividans wildtype (WT) strain and its co‐embedded mutant ΔK (lacking a functional kcsA gene) were conducted by labelling as the surface matrix of either one was labelled by cationic colloidal thorium dioxide. Within the WT strain, additional polyP was found to accumulate distinctly at the inner face of the cytoplasmic membrane. After removal of the cell wall (within protoplasts), the polyP‐derived lead‐sulphide (PbS) precipitate formed clusters of fibrillar material extending up to 50 nm into the cytoplasm. This feature was absent in the ΔK mutant strain. Together the results revealed that the presence of the KcsA channel and the structured polyP coincide.     

Peculiarities of imaging one- and two-dimensional structures using an electron microscope in the mirror operation mode

Measurements performed in an electron microscope with the mirror operation mode are most sensitive to local electric fields and geometrical roughness of any kind of the object being studied. The object with a geometrical relief is equivalent to a smooth surface with an effective distribution of microfields. Electrons forming the image interact with the local microfields for an extended time: during approach to the object, deceleration and acceleration away from the object. As a result, the electron trajectories can be strongly distorted, and the contrast changes essentially, leading to image deformation of details of the object under investigation and to lowering of the resolution. These effects are theoretically described and are illustrated by experiments. An analysis of these effects enables the real size and the shape of the object involved to be reconstructed.     

Immuno-EM using colloidal metal nanoparticles and electron spectroscopic imaging for co-localization at high spatial resolution

Multiple‐labelling immuno‐EM is a powerful tool for localizing and co‐localizing different antigens simultaneously in cells and tissues at high spatial resolution. Commonly used labels for this purpose are differently sized gold spheres. A comparison of results obtained with differently sized markers is often difficult, because the diameters of markers influence labelling efficiency. In the current study, we investigate a method for high‐resolution multiple‐labelling immuno‐EM, using equally sized colloidal markers made of different metals. Energy filtering transmission electron microscopy is used to differentiate particles based on elemental composition. The labels consist of colloidal gold, palladium and platinum‐core gold‐shell particles of approximately 6 nm in diameter, which are conjugated to different primary antibodies. Applicability of the electron spectroscopic imaging, methodology is demonstrated by labelling of actin, α‐actinin and myosin on ultra‐thin cryosections of skeletal muscle tissue.     

Peculiarities of imaging one- and two-dimensional structures in an emission electron microscope. 1. Theory

Local changes in work function cause deviations of the electrical microfield near a sample surface as a result of the uniform accelerating field distribution between the sample (cathode) and the extractor electrode (anode). This results in a change in the electron trajectories. As a consequence, the microscope image shows remarkable changes in position, size, intensity and lateral resolution of distinct details, which can be quantitatively described by the calculations presented here. Analysing these effects in the image gives an opportunity to determine the real lateral size of the observed structures and the distribution of local contact potentials.     

What atomic resolution annular dark field imaging can tell us about gold nanoparticles on (1 1 0)

Annular dark field scanning transmission electron microscopy imaging was recently applied to a catalyst consisting of gold nanoparticles on TiO₂ (1 1 0), showing directly that the gold atoms in small nanoparticles preferentially attach to specific sites on the TiO₂ (1 1 0) surface. Here, through simulation, a parameter exploration of the imaging conditions which maximise the visibility of such nanoparticles is presented. Aberration correction, finite source size and profile imaging are all considered while trying to extracting the maximum amount of information from a given sample. Comment is made on the role of the thermal vibration of the atoms in the nanoparticle, the magnitude of which is generally not known a priori but which affects the visibility of the nanoparticles in this imaging mode.     

Atomic resolution imaging of beryl: an investigation of the nano‐channel occupation

Beryl in different varieties (emerald, aquamarine, heliodor etc.) displays a wide range of colours that have fascinated humans throughout history. Beryl is a hexagonal cyclo‐silicate (ring‐silicate) with channels going through the crystal along the c‐axis. The channels are about 0.5 nm in diameter and can be occupied by water and alkali ions. Pure beryl (Be₃Al₂Si₆O₁₈) is colourless (variety goshenite). The characteristic colours are believed to be mainly generated through substitutions with metal atoms in the lattice. Which atoms that are substituted is still debated it has been proposed that metal ions may also be enclosed in the channels and that this can also contribute to the crystal colouring. So far spectroscopy studies have not been able to fully answer this. Here we present the first experiments using atomic resolution scanning transmission electron microscope imaging (STEM) to investigate the channel occupation in beryl. We present images of a natural beryl crystal (variety heliodor) from the Bin Thuan Province in Vietnam. The channel occupation can be visualized. Based on the image contrast in combination with ex situ element analysis we suggest that some or all of the atoms that are visible in the channels are Fe ions.     

The effect of deformation on the lateral resolution of atomic force microscopy

A computer model based on the elastic properties of rubber is introduced for the evaluation of the lateral resolution in atomic force microscopy of deformable specimens. The computational results show that, if the full width at half-height can be defined as the lateral resolution, it is continuously improved at greater probe forces, at the expense of a reduced molecular height. In fact, even for a probe that is bigger than the molecule, the real size of the molecule can be 'recovered' at about 25% compression. This result demonstrates that for a better lateral resolution, a greater probe force can be beneficial, provided that the molecule is not moved or damaged and the response remains elastic. Measurements on isolated low-density lipoproteins (LDL) show that with 26% vertical compression, the lateral size measured in atomic force microscopy is only about 72% of the value predicted by a simple convolution, and is only slightly larger (≈ 13%) than the known size of LDL. Therefore, the results on LDL provide a direct support for the conclusions of the computational model.     

Local estimation of lattice constants in HRTEM images

A model-based approach to estimate lattice constants from an atomically resolved HRTEM image is presented. The approach only utilizes the inherent periodicity of these images and does not require a centrosymmetric structure of the specimen. This allows the evaluation of, for instance, wurtzite-based materials like InGaN/GaN heterostructures. The lattice constants are determined within precisions below 3 pm from areas only a few unit cells large. This makes this method suitable for further strain/compositional analysis. Furthermore, the impact of the approximations of the true detector's covariance matrices on the assessment of the model-based approach is investigated, and insights into the quality of these noise models of the detector are gained.     

Combining accurate defocus with low-dose imaging in high resolution electron microscopy of biological material

High resolution (< 2 nm) electron microscopy of biological specimens requires three exacting conditions to be met simultaneously: (a) fine specimen detail must be protected from destruction by the electron beam (low dose), (b) the electron optics must be adjusted to be capable of imaging that detail interpretably (accurate defocus), and (c) a suitable field of interest must be identified. We describe a method encompassing all three with an 80% success rate using only minor modifications to a transmission electron microscope, and no expensive on-line computing.     

Studies of supported metal catalysts using high resolution secondary electron imaging in a STEM

An additional technique for use in the characterization of catalysts by electron microscopy is presented. High resolution secondary electron images obtained in a VG HB501 scanning transmission electron microscope have been used to study the surface topography of catalysts consisting of small metal particles on high surface area carbon supports. Surface features down to nanometre dimensions can be seen, allowing the examination of micropores in the support as well as larger pore structures. The results are compared with pore size distributions determined by gas adsorption methods, and are shown to yield valuable additional information. In addition, the method in principle allows examination of the locations of small metal catalyst particles on the support.