High resolution imaging properties of the stem

The effect of the finite size of the atom on the resolution of the STEM is investigated. When the probe size becomes comparable to the size of the atom, the quality of the image depends on the scattering properties of the atom as well as the distribution of electrons in the probe. A technique for calculating the image of a single atom is developed by expanding the scattering amplitude. This allows the image of an atom or its spatial frequency to be expanded into various components. The specific case of dark field contrast formed with elastically scattered electrons is considered. The coefficients of the components are evaluated for carbon and thorium using complex scattering amplitudes derived from relativistic Hartree-Fock-Slater wavefunctions. The coefficients are evaluated for a 100 keV microscope using an immersion type objective lens whose aperture is limited to 12 mrad by primary spherical aberration and a 100 keV microscope using the same objective lens in conjunction with a corrector lens for spherical aberration. Secondary spherical aberration limits the objective aperture of the corrected microscope to 30 mrad.     

High resolution SE-I SEM study of enamel crystal morphology

Until recently high resolution TEM was the only imaging mode capable of probing the atomic lattice structure of crystals composing tooth enamel. Studies designed to determine the polyhedral shape of normal enamel crystals and initiation of carious lesions in enamel crystals were hampered and limited by interpretation of two-dimensional TEM images from thin section and freeze fracture replica specimens lacking depth of field. The newly developed SE-I signal mode for SEM (SE-I/SE-II ratio) can produce images of enamel crystals approaching beam diameter dimensions (0.7–2.0 nm), rivaling the resolution of the TEM technique and generating topographic contrasts for three dimensional imaging at very high magnification (≈?1,000,000 X). Ultrathin chromium (Cr) films generate enriched high resolution SE-I contrasts of enamel crystal surfaces and when imaged using an immersion lens field emission SEM operated at high voltage (20–30 KeV) produce unsurpassed topographic contrasts. Since the grain size of Cr is below the resolution of any SEM and is ultrathin (≈?1 nm), then SE-I images can provide a more accurate representation of enamel crystal structure than TEM methodologies. Our SE-I SEM observations of normal human enamel crystals reveal fractured spicules which contain angled flat surfaces delineated by a prominent 2 nm wide SE-I edge brightness contrast. Although microscopic observations often show crystals which are hexagonal in cross-section, in both SEM and TEM many other growth habits, including rectangular or irregular crystals (30–40 nm in width) which contain “notches,” are also observed. More detailed morphological studies are therefore required to determine the most likely habit planes and their relevance to the function of the enamel crystals. The granular appearing fine structural contrast imposed onto <100> lattice planes of sectioned enamel in TEM micrographs is also resolved with topographic contrasts in SE-I micrographs. These granules probably represent one or both of the enamel protein classes.     

High resolution SEM imaging of gold nanoparticles in cells and tissues

The growing demand of gold nanoparticles in medical applications increases the need for simple and efficient characterization methods of the interaction between the nanoparticles and biological systems. Due to its nanometre resolution, modern scanning electron microscopy (SEM) offers straightforward visualization of metallic nanoparticles down to a few nanometre size, almost without any special preparation step. However, visualization of biological materials in SEM requires complicated preparation procedure, which is typically finished by metal coating needed to decrease charging artefacts and quick radiation damage of biomaterials in the course of SEM imaging. The finest conductive metal coating available is usually composed of a few nanometre size clusters, which are almost identical to the metal nanoparticles employed in medical applications. Therefore, SEM monitoring of metal nanoparticles within cells and tissues is incompatible with the conventional preparation methods. In this work, we show that charging artefacts related to non‐conductive biological specimen can be successfully eliminated by placing the uncoated biological sample on a conductive substrate. By growing the cells on glass pre‐coated with a chromium layer, we were able to observe the uptake of 10 nm gold nanoparticles inside uncoated and unstained macrophages and keratinocytes cells. Imaging in back scattered electrons allowed observation of gold nanoparticles located inside the cells, while imaging in secondary electron gave information on gold nanoparticles located on the surface of the cells. By mounting a skin cross‐section on an improved conductive holder, consisting of a silicon substrate coated with copper, we were able to observe penetration of gold nanoparticles of only 5 nm size through the skin barrier in an uncoated skin tissue. The described method offers a convenient modification in preparation procedure for biological samples to be analyzed in SEM. The method provides high conductivity without application of surface coating and requires less time and a reduced use of toxic chemicals.     

High resolution imaging by 1 MV electron microscopy

High voltage electron microscopy is well suited to the high resolution imaging of local structural changes in a material. New information at atomic resolution can be obtained on a broad range of material problems. Investigations of the structure of dislocations and planar faults in metallic and covalent crystals is presented.     

Atomic resolution ADF-STEM imaging of organic molecular crystal of halogenated copper phthalocyanine

Annular dark-field (ADF) scanning transmission electron microscopy (STEM) measurements are demonstrated for the first time to be applicable for acquiring Z-contrast images of organic molecules at atomic resolution. High-angle ADF imaging by STEM is a new technique that provides incoherent high-resolution Z-contrast images for organic molecules. In the present study, low-angle ADF-STEM is successfully employed to image the molecular crystal structure of hexadecachloro-Cu-phthalocyanine (Cl₁₆-CuPc), an organic molecule. The structures of CuPc derivatives (polyhalogenated CuPc with Br and Cl) are determined quantitatively using the same technique to determine the occupancy of halogens at each chemical site. By comparing the image contrasts of atomic columns, the occupancy of Br is found to be ca. 56% at the inner position, slightly higher than that for random substitution and in good agreement with previous TEM results.     

High resolution imaging of surface patterns of single bacterial cells

We systematically studied the origin of surface patterns observed on single Sinorhizobium meliloti bacterial cells by comparing the complementary techniques atomic force microscopy (AFM) and scanning electron microscopy (SEM). Conditions ranged from living bacteria in liquid to fixed bacteria in high vacuum. Stepwise, we applied different sample modifications (fixation, drying, metal coating, etc.) and characterized the observed surface patterns. A detailed analysis revealed that the surface structure with wrinkled protrusions in SEM images were not generated de novo but most likely evolved from similar and naturally present structures on the surface of living bacteria. The influence of osmotic stress to the surface structure of living cells was evaluated and also the contribution of exopolysaccharide and lipopolysaccharide (LPS) by imaging two mutant strains of the bacterium under native conditions. AFM images of living bacteria in culture medium exhibited surface structures of the size of single proteins emphasizing the usefulness of AFM for high resolution cell imaging.     

High resolution imaging of defects in sigma phase in a stainless steel

The high resolution axial lattice imaging technique has been applied to the study of the faults which commonly occur in the sigma phase which can be formed from ferrite in a duplex high chromium stainless steel on ageing at 973 K. It was noted that axial lattice fringe images at (001)_σ show no detailed correspondences with thickness and it was further demonstrated that lattice fringe displacements in axial images at strain free boundaries can be used to give an accurate measure of the magnitude of the associated lattice displacements. The displacement vectors associated with the common (100)_σ faults in the material were classified.     

High spatial resolution spectroscopy in the elemental microanalysis and imaging of biological systems

The application of analytical electron microscopy to the high spatial resolution study of biological systems is reviewed. Specimen preparation, quantitative analysis, capabilities and limitations are all discussed, principally in the context of energy-dispersive X-ray analysis. Results are presented using both current techniques and the developing quantitative image analysis. Finally the role of new instrumental approaches, including electron energy loss spectrometry, is discussed.     

Bioimaging TOF-SIMS: High resolution 3D imaging of single cells

The distribution of phosphocholine ions (m/z 184, m/z 86), sodium ions, and potassium ions in thyroid tumor cells was analyzed by imaging TOF-SIMS. Repeated sputtering with a C(60) (+) source and subsequent analysis with a Bi(3) (+) gun produced a series of 138 images that were stacked to make a 3D display of the chemistry of cells. Phosphocholine was seen in the plasma membrane (m/z 184) and intracellular membranes (m/z 86). The different fragmentation of the phospholipid probably reflects the chemical composition of membranes at these sites. High intensity of secondary ion signals of potassium was seen in membrane-encompassed cellular compartments. The data indicate that potassium ions are compartmentalized in thyroid tumor cells.     

Problems of interpretation in high resolution electron microscopy

Current assumptions in wave aberration theory and specimen scattering theory are reviewed. More quantitative image simulations would be valuable as well as use of a wider range of imaging techniques, particularly STEM. The severe difficulties of high resolution three-dimensional reconstruction are described and illustrated.     

High resolution non-contact AFM imaging of liquids condensed onto chemically nanopatterned surfaces

The wetting of ethanol and octane on chemically nanopatterned surfaces has been investigated using Atomic Force Microscopy (AFM) under controlled environmental conditions. The patterns were generated on a methyl-terminated, organic monolayer using an AFM electro-oxidation process. The subsequent wetting of the organic liquids was studied using non-contact mode AFM under equilibrium conditions with the vapor. This study of condensed nanoliquids provides the first reliable measurements of sub 100 nm liquid profile shapes. The derived contact angles give an estimate of the line tension.     

High spatial resolution secondary ion imaging and secondary ion mass spectrometry of aluminium-lithium alloys

Samples of aluminium-lithium alloys have been observed by scanning ion microscopy and analysed by secondary ion mass spectrometry. The high signal-to-noise ratio of the positive secondary lithium ion opens up the possibility of both high resolution imaging and microanalysis of lithium distributions in aluminium and other materials. Some of the problems encountered due to sample preparation are discussed and ion images of both the artefacts and the true lithium distribution are shown.     

Element specific imaging with high lateral resolution: an experimental study on layer structures

Planar defects and individual layers in ceramic material are chemically imaged by high resolution energy-filtering TEM using a post-column imaging electron energy filter. Objects are barium layers in the cuprate superconductor NdBa₂Cu₃O_7−δ (isostructual to YBa₂Cu₃O_7−δ) as well as planar defects and precipitates of β-WB in tungsten- and chromium-doped TiB₂. The barium layers with a spacing of 0.42 nm in the cuprate are resolved in jump-ratio images using the Ba_N edge. In the boride system the β-WB precipitates with thickness of 0.8 nm can be chemically imaged in elemental maps of B_K, Ti_L ,Cr_L and W_M. The B as well as the Ti map show a decrease in intensity at the precipitates, whereas in the W map an increase in intensity is observed. The boron-deficient layers with a spacing of 0.38 nm in the β-WB precipitate can be resolved in boron jump-ratio images. Additionally, defects containing single boron-deficient layers are chemically imaged. Hence structures in the dimension of interatomic distances can be imaged with respect to their elemental constituents. Although high resolution electron spectroscopic images contain strong interference contrast from elastic scattering, after normalization or background subtraction the element specific images are dominated by chemical contrast.     

Comparative study on the reachable workspaces of regular and irregular axially symmetric hexapod robots

Journal of Mechanical Science and Technology - Six-legged hexapod walking robots are well-known for their intrinsic stability during navigation and 6-DoF object manipulation. The robot must be...     

High resolution X-ray phase contrast synchrotron imaging of normal and ligation damaged rat sciatic nerves

This study was performed to apply synchrotron radiation (SR) imaging to a neuropathologic evaluation technique after treatment of peripheral nerve blocks. A phase contrast synchrotron images of normal and ligation damaged rat sciatic nerve were obtained with an 8 KeV monochromatic beam and 20-mum thick CsI(TI) scintillation crystal. The visual image was magnified using a 20x microscope objective and captured using an analog CCD camera. Obtained images were compared with conventional light microscopic findings from the same nerve samples. By using an edge enhancement effect of phase const with SR, we could easily discriminate each nerve fiber and identify the arrangement of nerve fibers within a whole thickness (about 1 mm in diameter) of peripheral nerve without sectioning and fixation. The composite SR image of a ligation damaged rat sciatic nerve sample showed that the response to nerve injury was different on each side of the site of injury. The SR image of damaged distal lesion showed destruction of neural microarchitecture and typical extensive Wallerian degeneration of nerve fibers as clearly as histologic image. We could get very detailed morphologic data for Wallerian degeneration of nerve fibers by using the SR imaging technique. We believe that the phase contrast synchrotron imaging has great potential as an imaging tool in the bioscience and medical science.     

High resolution absorption spectroscopy of exploding wire plasmas using an x-pinch x-ray source and spherically bent crystal

We present here the use of absorption spectroscopy of the continuum radiation from x-pinch-produced point x-ray sources as a diagnostic to investigate the properties of aluminum plasmas created by pulsed power machines. This technique is being developed to determine the charge state, temperature, and density as a function of time and space under conditions that are inaccessible to x-ray emission spectroscopic diagnostics. The apparatus and its characterization are described, and the spectrometer dispersion, magnification, and resolution are calculated and compared with experimental results. Spectral resolution of about 5000 and spatial resolution of about 20 μm are demonstrated. This spectral resolution is the highest available to date in an absorption experiment. The beneficial properties of the x-pinch x-ray source as the backlighter for this diagnostic are the small source size (<5 μm), smooth continuum radiation, and short pulse duration (<0.1 ns). Results from a closely spaced (1 mm) exploding wire pair are shown and the general features are discussed.     

Elemental imaging and resolution in energy-filtered conventional electron microscopy

Energy-filtered transmission electron microscopy (EFEM) was used to image the distributions of uranium and carbon in uranyl acetate stained catalase crystals. The spatial resolution obtained from inelastic C K-edge and U O4,5-edge images, determined from the highest-order reflection in the computed diffraction pattern, was 3.4 nm for both carbon and uranium. The resolution limit imposed by the delocalization of inelastic scattering was estimated from cross-section measurements to be 0.6 nm for U and 0.2 nm for C. Considering both delocalization and the effects of microscope aberrations, for an objective lens chromatic aberration coefficient of 2.8 mm and 10 eV energy window, the calculated resolutions are 2.0 nm for C and 1.2 nm for U. The effects of plural inelastic and elastic-inelastic scattering were sufficiently large to show crystalline structure in unprocessed pre-edge inelastic images. Previously suggested methods for eliminating these artifacts were applied to obtain the compositional information in the catalase EFEM images.     

High resolution imaging of the CdTe/(100)InSb interface: A lattice-matched hetero-epitaxial structure

This paper reviews techniques available for obtaining images which can distinguish the two components in lattice-matched, hetero-epitaxial structures. From series of computer simulations it is shown that in the case of CdTe/(100)InSb the usual criteria for ‘good’ high resolution imaging (i.e. thin crystal, Scherzer focus) do not give images which differentiate the two materials. Conditions which do show different contrast features for both CdTe and InSb are found; under these conditions images are obtained which reveal the interfacial structure clearly, and show it to be essentially flat, and free of defects within the regions examined. Prospects for obtaining true ‘atomic’ images using a new 400 kV HREM are outlined.