Standard Transmission Electron Microscopy Research Articles

Versatile application of a modern scanning electron microscope for materials characterization

Scanning electron microscopy (SEM) is an indispensable characterization technique for materials science. More recently, scanning electron microscopes can be equipped with scanning transmission electron microscopy (STEM) detectors, which considerably extend their capabilities. It is demonstrated in this work that the correlative application of SEM and STEM imaging techniques provides comprehensive sample information on nanomaterials. This is highlighted by the use of a modern scanning electron microscope, which is equipped with in-lens and in-column detectors, a double-tilt holder for electron transparent specimens and a CCD camera for the acquisition of on-axis diffraction patterns. Using multi-walled carbon nanotubes and Pt/Al2O3 powder samples we will show that a complete characterization can be achieved by combining STEM (mass-thickness and diffraction) contrast and SEM (topography and materials) contrast. This is not possible in a standard transmission electron microscope where topography information cannot be routinely obtained. We also exploit the large tilt angle range of the specimen holder to perform 180 degrees STEM tomography on multi-walled carbon nanotubes, which avoids the missing wedge artifacts.

CLEM with Commercially Available Reagents to Facilitate Immunolocalization

Abstract:

Sequential plan-view imaging of sub-surface structures in the transmission electron microscope

Transmission electron microscopy (TEM) is a central technique for the characterisation of materials at the atomic scale. However, it requires the sample to be thin enough to be electron transparent, imposing strict limitations when studying thick structures in plan-view. Here we present a method for sequential plan-view TEM that allows one to image complex structures at various depths. The approach consists of performing an iterative series of front-side ion milling followed by TEM imaging. We show it is possible to image how the sample properties vary with depth up to several microns below the surface, with no degradation of the sample and imaging conditions throughout the experiment. We apply this approach to 3D cavities in mesoporous GaN distributed Bragg reflectors, demonstrating the ability to characterise the morphology of the pores, local crystal features and chemical composition through the multilayer structure. The same workflow can be applied to a variety of complex micron-scale systems which are by nature too thick for standard TEM analysis, and can also be adapted for profiling samples in cross-section.

Observing the Quantum Wave Nature of Free Electrons through Spontaneous Emission.

We investigate, both experimentally and theoretically, the interpretation of the free-electron wave function using spontaneous emission. We use a transversely wide single-electron wave function to describe the spatial extent of transverse coherence of an electron beam in a standard transmission electron microscope. When the electron beam passes next to a metallic grating, spontaneous Smith-Purcell radiation is emitted. We then examine the effect of the electron wave function transversal size on the emitted radiation. Two interpretations widely used in the literature are considered: (1)radiation by a continuous current density attributed to the quantum probability current, equivalent to the spreading of the electron charge continuously over space; and (2)interpreting the square modulus of the wave function as a probability distribution of finding a point particle at a certain location, wherein the electron charge is always localized in space. We discuss how these two interpretations give contradictory predictions for the radiation pattern in our experiment, comparing the emission from narrow and wide wave functions with respect to the emitted radiation's wavelength. Matching our experiment with a new quantum-electrodynamics derivation, we conclude that the measurements can be explained by the probability distribution approach wherein the electron interacts with the grating as a classical point charge. Our findings clarify the transition between the classical and quantum regimes and shed light on the mechanisms that take part in general light-matter interactions.

Controlling the size and shape of liposomal ciprofloxacin nanocrystals by varying the lipid bilayer composition and drug to lipid ratio

Drug nanocrystals precipitated inside liposomes are of increasing interest in liposomal drug delivery. For liposomal nanocrystal formulations, the size and shape of the drug nanocrystals can influence the apparent drug release properties, providing opportunities for developing tailored liposomal drug release systems. Small angle X-ray scattering (SAXS) and quantitative transmission electron microscopy (TEM) can be used to analyse the size distributions of the nanoparticles. In this study, by changing the fluidity of the membrane through the use of different membrane phospholipids with varying cholesterol content, the impact of lipid phase, fluidity and permeability on the size distribution of ciprofloxacin nanocrystals were investigated using standard TEM and SAXS as orthogonal techniques. The results show that the phospholipid phase behaviour has a direct effect on the nanocrystal size distribution, where shorter and thinner nanocrystals were formed in liposomes made from hydrogenated soy phosphatidylcholine (HSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) phospholipids with higher phase transition temperatures than 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with lower transition temperatures. This is mainly due to the phase behaviour of the liposome during nanocrystal formation. The addition of cholesterol that reduces fluidity and permeability of the DOPC liposomes was also shown to restrict the growth of the ciprofloxacin nanocrystals. Moreover, increasing the drug loading of the liposomes made from HSPC and DPPC produced longer and wider nanocrystals. The findings open new opportunities to tailor nanocrystal size distributions, as well as the aspect ratio of the enclosing liposomes with potential to alter drug release and in vivo behaviour.

Forescattered electron imaging of nanoparticles in scanning electron microscopy

In this study, we have used a Zr-Nb alloy containing well-defined nano-precipitates as a model material in which to study imaging contrast inversions (atomic number or diffraction contrast) observed with the forescattered electron imaging system, ARGUS™, in a scanning electron microscope (SEM) when imaging a thin foil in a transmission geometry. The study is based on Monte Carlo simulations and analysis of micrographs experimentally acquired under different imaging conditions. Based on the results, imaging conditions that enhance atomic number or diffraction contrast have been proposed. Data acquired from the ARGUS™ imaging system in an SEM has also been compared with results from standard transmission electron microscopy and scanning transmission electron microscopy imaging of the same material. These results demonstrate the capability of the ARGUS™ system to investigate microstructures in nano-scale materials.

Influence of ethanol blending ratios on in-flame soot particle structures in an optical spark-ignition direct-injection engine

This study shows how ethanol addition impacts in-flame and exhaust soot in spark-ignition direct injection (SIDI) engines through direct and simultaneous sampling of particles from the flames and exhaust stream. The thermophoresis-based soot sampling and transmission electron microscope (TEM) imaging was performed for various ethanol blending ratios ranging from 0 to 60%. A quantitative analysis is performed in the images obtained from a standard TEM by yielding the in-flame and exhaust soot number counts, projection area, as well as morphological parameters such as soot primary particle diameter, aggregate radius of gyration, and fractal dimension. In addition, the internal structures and reactivity status of in-flame and exhaust soot particles are unveiled using a high-resolution TEM with carbon fringe length, tortuosity and fringe-to-fringe separation extracted from processed carbon layer fringe images. The results show that both the number counts and projection areas of exhaust soot are much lower than those of the in-flame soot at any fixed ethanol blending ratios. The exhaust soot is smaller in size for both the primary particles and aggregates with more compact aggregate structures, longer and straighter fringe, and lower fringe separation, all indicating significant soot oxidation occurred inside the cylinder before the particles exit through the exhaust. With increasing ethanol blending ratio, almost linear reduction in number counts and projection area is found for both the in-flame and exhaust soot particles, while both soot aggregates and primary particles become smaller with more stretched aggregate structures and shorter, more curved, and narrower gap fringe structures, indicating soot particles with higher reactivity. The significantly lower soot emissions achieved with higher ethanol blending ratio is found to be due primarily to the suppression of soot formation within the flame as evidenced by higher reduction rate of number counts, projection area, and primary particle and aggregate sizes for the in-flame soot than that of the exhaust soot.

Dielectric, magnetic and photocatalytic activity of PolyPyrrole/Prussian red nanocomposite for waste water treatment applications

This work describes the synthesis and characterization of a novel nanocomposite of Prussian red and polypyrrole using standard protocols (Fourier-transform infrared spectroscopy, powder X-ray diffraction (XRD) and Transmission electron microscopy analysis). The comparative studies of the synthesized nanocomposite, pristine polypyrrole and related (PPY) nanocomposites with other dopants indicate an enhancement in the thermal, mechanical and electrical properties over pure {(PPY) and related PPY nanocomposites}.The investigated physicochemical properties of nanocomposite suggest desirable material properties for environmental applications. The synthesized nanocomposite was observed to have a transition from paramagnetic to ferromagnetic behavior with a decreasing temperature and also displayed good photocatalytic activity towards dye and antibiotic degradation. The dye degradation of more than 80% under the pseudo-first order conditions as observed for, Methyl orange Methylene blue and Rhodamine B dyes in 80 min at room temperature and neutral pH. Besides, it showed photocatalytic activity towards the degradation of antibiotics: rifampicin, levofloxacin and amoxicillin with good results. Furthermore, the nanocomposite displayed selective adsorption capacity towards recovery of precious metal ions in the waste water.

The Effect of Apocynin on Motor and Cognitive Functions in Experimental Alzheimer’s disease

Scope: We investigated the potential beneficial effect of Apocynin (APO) on motor and cognitive functions in experimental Alzheimer’s disease (AD). Materials and Methods: Experimental AD was induced in rats by intraventricular streptozotocin (STZ) injection. Sham group received artificial cerebrospinal fluid (CSF). Both groups were randomly divided into two subgroups. One of the subgroups received intraperitoneal APO for while the other had normal saline (NS). The animals were evaluated with rotarod, accelerod and Water-Maze tests before and after the treatment. Additionally, biochemical markers of oxidative stress such as malondialdehyde (MDA) and reduced glutathione (GSH) were analyzed from brain specimens. Standard histological evaluation and transmission electron microscopy (TEM) were used to evaluate the neural damage. Results: The difference between STZ+NS in comparison with CSF+NS, CSF+APO and STZ+APO were statistically significant on 30 and 40 rpm on rotarod test. GSH levels, accelerod and Water-Maze test results were not statistically significant between subgroups. However, MDA differences between STZ+NS in comparison with CSF+NS, CSF+APO and STZ+APO were statistically significant. Hemotoxilene eozine staining and TEM results showed apocynins protective effect. Conclusion: These results indicate that APO can provide neuro-protective effect for motor but not for cognitive performance in experimental AD. Keywords: Alzheimer’s disease, Streptozotocin, Apocynin, Rotarod test, Accelerod test, Water-Maze test

Diagnosis of Primary Ciliary Dyskinesia. An Official American Thoracic Society Clinical Practice Guideline.

This document presents the American Thoracic Society clinical practice guidelines for the diagnosis of primary ciliary dyskinesia (PCD). Clinicians investigating adult and pediatric patients for possible PCD. Systematic reviews and, when appropriate, meta-analyses were conducted to summarize all available evidence pertinent to our clinical questions. Evidence was assessed using the GRADE (Grading of Recommendations, Assessment, Development and Evaluation) approach for diagnosis and discussed by a multidisciplinary panel with expertise in PCD. Predetermined conflict-of-interest management strategies were applied, and recommendations were formulated, written, and graded exclusively by the nonconflicted panelists. Three conflicted individuals were also prohibited from writing, editing, or providing feedback on the relevant sections of the manuscript. After considering diagnostic test accuracy, confidence in the estimates for each diagnostic test, relative importance of test results studied, desirable and undesirable direct consequences of each diagnostic test, downstream consequences of each diagnostic test result, patient values and preferences, costs, feasibility, acceptability, and implications for health equity, the panel made recommendations for or against the use of specific diagnostic tests as compared with using the current reference standard (transmission electron microscopy and/or genetic testing) for the diagnosis of PCD. The panel formulated and provided a rationale for the direction as well as for the strength of each recommendation to establish the diagnosis of PCD.

In English

Weakly acidic cation exchange resin was modified with nanoparticles of zirconium hydrophosphate. The materials were investigated with methods of standard contact porosimetry and transmission electron microscopy. Both non-aggregated nanoparticles (4–15 nm) and larger formations (from 250 nm to several microns) have been found. Single particles in clusters and channels of the polymer depress dissociation of carboxylic groups due to counter-ions (H + ) in electric double layer around the particles. This causes transformation of porous structure of the polymer: the contribution of micropores to total porosity increases. These additional selective sites provide stronger interaction of Brilliant Green molecules with the surface in comparison with the pristine polymer. Removal of the dye from deionized water and Ni(II) ions from water containing also hardness ions was studied under dynamic conditions. The composite shows higher break-through capacity than that of the pristine resin. The modifier also facilitates regeneration of the weakly acidic ion-exchanger.

Pattern of collagen fibers and localization of matrix metalloproteinase 2 and 9 during breast cancer invasion.

Parenchymal cells naturally interact, react and adapt with the environment including stromal components around them in order to maintain tissue architecture and function. However, studies have shown that this spontaneous interaction will become crucial in assisting cancer invasion. The purpose of the study was to analyze the pattern of collagen deposition and localization of matrix metalloproteinase 2 and matrix metalloproteinase 9 in the tumor microenvironment during breast cancer invasion. A standard transmission electron microscopy procedure was used together with the immunogold technique with a few modifications. The ultrastructure of fibroblasts in the vicinity of cancer cells was thick, elongated and spindle shaped with nuclear indentations. Desmoplasia was present near the cancer cells. Collagen fibers were still arranged parallel to the cancer cells and fibroblasts but were less dense than collagen fibers far from cancer cells and fibroblasts. Collagen fibers were less dense in the pericellular region because of proteolytic enzyme activity, which facilitates the invasion of breast cancer cells. In immunogold localization analysis, matrix metalloproteinase 9 had consistent localization throughout cancer cells, fibroblast and stroma. In matrix metalloproteinase 2 localization, gold conjugates were more heavily deposited in cancer cells and fibroblasts than in the stroma. Invasive breast carcinoma is not an independent entity, and its survival depends on the surrounding microenvironment.

Towards 3D crystal orientation reconstruction using automated crystal orientation mapping transmission electron microscopy (ACOM-TEM).

To relate the internal structure of a volume (crystallite and phase boundaries) to properties (electrical, magnetic, mechanical, thermal), a full 3D reconstruction in combination with in situ testing is desirable. In situ testing allows the crystallographic changes in a material to be followed by tracking and comparing the individual crystals and phases. Standard transmission electron microscopy (TEM) delivers a projection image through the 3D volume of an electron-transparent TEM sample lamella. Only with the help of a dedicated TEM tomography sample holder is an accurate 3D reconstruction of the TEM lamella currently possible. 2D crystal orientation mapping has become a standard method for crystal orientation and phase determination while 3D crystal orientation mapping have been reported only a few times. The combination of in situ testing with 3D crystal orientation mapping remains a challenge in terms of stability and accuracy. Here, we outline a method to 3D reconstruct the crystal orientation from a superimposed diffraction pattern of overlapping crystals without sample tilt. Avoiding the typically required tilt series for 3D reconstruction enables not only faster in situ tests but also opens the possibility for more stable and more accurate in situ mechanical testing. The approach laid out here should serve as an inspiration for further research and does not make a claim to be complete.

Radiation Damage Mechanisms of Monolayer-Protected Nanoparticles via TEM Analysis

In this work, it is shown that thiol-protected Au nanoparticles (AuNPs@SR) of approximately 3.4 nm size suffered unexpectedly high radiation damage under standard transmission electron microscopy (TEM) operating conditions. For metallic systems (conducting sample), it is expected that the greatest contribution to the damage comes from knock-on displacement, but radiolysis is the most probable radiation damage mechanism for organic samples. The radiation damage of the electron beam produces huge changes in AuNPs’ structure, leading to coalescence of the Au cores when their {100} surfaces are facing each other. The complete coalescence process involve thiol desoprtion, AuNPs’ reorientation, and surface diffusion of Au adatoms, which produce the oriented attachment of the Au cores. The knock-on displacement cannot explain by itself the time taken by the entire process. Through a rigorous analysis, we rationalize the results considering that because of the small size of AuNPs they have a lower electron densit...

Fine structure of the foraminifer Haynesina germanica (Ehrenberg) and its sequestered chloroplasts

Abstract The common, shallow-water foraminiferan Haynesina germanica (Ehrenberg) is one of a growing number of benthic rotalids known to acquire phototrophy through chloroplast retention (kleptoplasty). This species is broadly distributed, tolerant to a range of environmental conditions, and has been identified as a potential bioindicator of moderately polluted settings. This study was undertaken to better understand the fine structural relationships of this foraminiferan and its sequestered chloroplasts. Individuals were selected from fresh field collections and prepared for transmission electron microscopy (TEM) using high pressure freezing followed by freeze substitution (HPF/FS). This method is well-known for its high-resolution of cellular structure without many of the potential artifacts introduced by standard TEM chemical fixation. Features of the sequestered chloroplasts are consistent with those of diatoms, and pennate diatoms are the most likely source. The sequestered chloroplasts observed in H. germanica are surrounded by five membranes: the four chloroplast membranes of the diatom and an encompassing, outer vacuolar membrane of the foraminifer. Most of the chloroplast-containing vacuoles include a single chloroplast, but some contain up to three. The chloroplasts examined have numerous lamellae, each consisting of three thylakoids. The pyrenoid is traversed by a single lamella that contains a single thylakoid. Storage granules are present in some chloroplasts. Food vacuoles, common in many smaller benthic foraminifera, are rare in this species, which is consistent with its utilization of acquired phototrophy as documented by others. Both uninucleate (typically a gamont) and multinucleate (typically an agamont) individuals were observed, suggesting that this species has an alternation of generations like most benthic foraminifera. Other features of the cell body (e.g., abundant mitochondria, active Golgi bodies, multivesicular bodies, peroxisomes) are consistent with its occurrences in shallow-water, aerobic settings. In addition, fibrillar vesicles are common as are small pyriform electron-dense bodies of unknown origin or function. Vesicles that appear to accumulate wastes are also common.

In-situ TEM mechanical characterization of nanowire in atomic scale using MEMS device

This paper presents an experimental method for atomic-scale observation of in situ transmission electron microscopy (TEM) tensile test using micro-electromechanical systems (MEMS) actuator. A carefully designed and fabricated thermal actuator was applied in a standard double-tilt TEM holder to provide precisely controllable stretching process. To demonstrate the capabilities of the tensile testing system, some copper nanowires were stretched under a slowly force loading and in situ atomic resolution images were achieved. In addition, some faults releasing processes were observed during the tensile stretching and the corresponding strain–stress curve was calculated. The implemented actuator for atomic-scale observation can potentially have broad application to studies of nanoscale materials.

In situ monitoring of dislocation proliferation during plastic deformation using ultrasound

Ultrasound has long been used as a non-destructive tool to test for the brittle fracture of materials. Could it be used as a similar tool to test for ductile failure? As a first step towards answering this question, we report results of local measurements of the speed of transverse waves in aluminum under standard testing conditions at two different probe locations and continuously as a function of applied load. The result, as expected, is independent of stress in the elastic regime, but there is a clear change, consistent with a proliferation of dislocations, as soon as the yield strength is reached. We use a model that blames the change in wave speed on the interaction of elastic waves with oscillating dislocation segments, which quantitatively relates the change in wave velocity with dislocation density Λ and segment length L, thus obtaining a continuous relation between dislocation density and externally applied stress. We took off samples from the probe before, at intermediate, and high loading, and we measured their dislocation density using standard X-ray diffraction and transmission electron microscopy techniques. The results agree well with the acoustic measurements, and the relation between stress and dislocation density is consistent with the Taylor rule. This indicates that monitoring the speed of transverse waves could become a useful diagnostic of dislocation density for metallic pieces in service as well as a tool to test models of plastic behavior.

A coarse-grained Monte Carlo approach to diffusion processes in metallic nanoparticles

A kinetic Monte Carlo approach on a coarse-grained lattice is developed for the simulation of surface diffusion processes of Ni, Pd and Au structures with diameters in the range of a few nanometers. Intensity information obtained via standard two-dimensional transmission electron microscopy imaging techniques is used to create three-dimensional structure models as input for a cellular automaton. A series of update rules based on reaction kinetics is defined to allow for a stepwise evolution in time with the aim to simulate surface diffusion phenomena such as Rayleigh breakup and surface wetting. The material flow, in our case represented by the hopping of discrete portions of metal on a given grid, is driven by the attempt to minimize the surface energy, which can be achieved by maximizing the number of filled neighbor cells.Graphical abstract

Atom size electron vortex beams with selectable orbital angular momentum

The decreasing size of modern functional magnetic materials and devices cause a steadily increasing demand for high resolution quantitative magnetic characterization. Transmission electron microscopy (TEM) based measurements of the electron energy-loss magnetic chiral dichroism (EMCD) may serve as the needed experimental tool. To this end, we present a reliable and robust electron-optical setup that generates and controls user-selectable single state electron vortex beams with defined orbital angular momenta. Our set-up is based on a standard high-resolution scanning TEM with probe aberration corrector, to which we added a vortex generating fork aperture and a miniaturized aperture for vortex selection. We demonstrate that atom size probes can be formed from these electron vortices and that they can be used for atomic resolution structural and spectroscopic imaging – both of which are prerequisites for future atomic EMCD investigations.

3D shaping of electron beams using amplitude masks

Shaping the electron wavefunction in three dimensions may prove to be an indispensable tool for research involving atomic-sized particle trapping, manipulation, and synthesis. We utilize computer-generated holograms to sculpt electron wavefunctions in a standard transmission electron microscope in 3D, and demonstrate the formation of electron beams exhibiting high intensity along specific trajectories as well as shaping the beam into a 3D lattice of hot-spots. The concepts presented here are similar to those used in light optics for trapping and tweezing of particles, but at atomic scale resolutions.