Transmission Electron Microscopy Sample Research Articles

A Hierarchical Nanostructure‐Based Surface‐Enhanced Raman Scattering Sensor for Preconcentration and Detection of Antibiotic Pollutants

A surface‐enhanced Raman scattering (SERS) sensor that integrates a plasmonic nanostructure with an electrostatic preconcentration unit has been developed. The silver nanoparticles (Ag‐NPs) grafted Ge nanowires (Ge‐NWs) are grown onto the surface of a commercial copper grid (Cu‐grid), which is usually used for transmission electron microscopy sample support. This leads to the formation of a hierarchical Ag‐NPs@Ge‐NWs@Cu‐grid substrate, which plays dual roles, i.e., preconcentration of analytes and amplification of SERS signal. This SERS detection platform based on the Ag‐NPs@Ge‐NWs@Cu‐grid substrate is able to selectively detect polar analytes such as antibiotic pollutants. This SERS sensor has achieved a limit of detection of 2.4 × 10−9m for 6‐aminopenicillanic acid and 0.9 × 10−9m for penicillin G, respectively. This work has demonstrated a promising SERS detection platform for monitoring polar SERS‐active pollutants in the aquatic environment.

A developed wedge fixtures assisted high precision TEM samples pre-thinning method: Towards the batch lamella preparation

Ion milling, wedge cutting or polishing, and focused ion beam (FIB) milling are widely-used techniques for the transmission electron microscope (TEM) sample preparation. Especially, the FIB milling provides a site-specific analysis, deposition, and ablation of materials in the micrometer and nanometer scale. However, the cost of FIB tools has been always a significant concern. Since it is inevitable to use the FIB technique, the improvement of efficiency is a key point. Traditional TEM sample preparation with FIB was routinely implemented on a single sample each time. Aiming at cost efficiency, a new pre-thinning technique for batch sample preparation was developed in this paper. The present proposal combines the sample preparation techniques with multi-samples thinning, cross-section scanning electron microscopy (SEM), wedge cutting, FIB and other sample pre-thinning techniques. The new pre-thinning technique is to prepare an edge TEM sample on a grinding and polishing fixture with a slant surface. The thickness of the wedges sample can be measured to 1∼2 μm under optical microscope. Therefore, this fixture is superior to the traditional optical method of estimating the membrane thickness. Moreover, by utilizing a multi-sample holding fixture, more samples can be pre-thinned simultaneously, which significantly improved the productivity of TEM sample preparation.

Mitigating Curtaining Artifacts During Ga FIB TEM Lamella Preparation of a 14 nm FinFET Device.

We report on the mitigation of curtaining artifacts during transmission electron microscopy (TEM) lamella preparation by means of a modified ion beam milling approach, which involves altering the incident angle of the Ga ions by rocking of the sample on a special stage. We applied this technique to TEM sample preparation of a state-of-the-art integrated circuit based on a 14-nm technology node. Site-specific lamellae with a thickness <15 nm were prepared by top-down Ga focused ion beam polishing through upper metal contacts. The lamellae were analyzed by means of high-resolution TEM, which showed a clear transistor structure and confirmed minimal curtaining artifacts. The results are compared with a standard inverted thinning preparation technique.

Microstructural evolution during tension-compression in-plane deformation of a pure aluminum sheet

Classically, the Bauschinger effect refers to a reduction of yield strength after a load path change. In this contribution, we present results of an experimental and microstructural investigation on Bauschinger effects in an AA1050 sheet metal (with 1 mm thickness) subjected to in-plane uniaxial loading. We performed tension-compression tests with different values of maximum tensile strains in a novel tool that was specifically designed to avoid buckling under compressive loading. Our experimental results show that the sheet material exhibits distinct Bauschinger effects. At different stages of deformation, we interrupted the tests and prepared samples for transmission electron microscopy. Our microstructural observations allow rationalizing the occurrence and magnitude of the observed Bauschinger effects.

Comparisons of methods to obtain insoluble particles in snow for transmission electron microscopy

Most studies of insoluble particles in snow have been focused on their mass concentration. Little is understood about the physicochemical properties of individual insoluble particles in snow. However, the information is essential to trace sources of the particles, to understand ice nuclei, and to quantify critical aerosol particles (e.g., black carbon) in snow analyzed by bulk methods. The lack of individual particle analyses of snow meltwater stems from the difficulty of producing feasible samples of the snow-borne insoluble particles. In this study, we examined six sample preparation methods and compared their results using transmission electron microscopy (TEM). The results are the following: (1) Drop-by-drop method (DDM) is the easiest method to make TEM samples but cannot remove the influence of the dissolved substances in snow meltwater. (2) Direct filtration method (DFM) was infeasible because the water penetration of carbon film on copper TEM grids is low. (3) Filtration and transfer method (FTM) is through using ultrasonication to transfer insoluble particles on the nuclepore polycarbonate membranes to TEM grids. The drawback of this method is that ultrasonication breaks individual particles into fragments. (4) Freeze-drying method (FDM) can result in new particles from the drying dissolved substances, which interferes with the identification of insoluble particles. (5) Dilution-gravity separation method (DGM) can obtain different substances based on their specific gravity in long standing water. The method can effectively reduce soluble substances but lose insoluble carbonaceous particles (e.g., soot and organic particles). (6) Tangential flow filtration and dilution (TFF-D) through concentrating and desalting dissolved substances is to remove the dissolved substances but keep insoluble particles in snow meltwater. The TFF-D method not only can be suitable for electron microscopy to study individual insoluble particles in snow meltwater but also for any offline microscopic observation such as Raman spectroscopy and mass spectrometry.

The terrestrial green macroalga Prasiola calophylla (Trebouxiophyceae, Chlorophyta): ecophysiological performance under water-limiting conditions

The phylogenetic placement of Prasiola calophylla, from an anthropogenic habitat previously shown to contain a novel UV sunscreen compound, was confirmed by analysis of its rbcL gene. This alga has the capacity to tolerate strong water-limiting conditions. The photosynthetic performance and ultrastructural changes under desiccation and osmotic stress were investigated. Freshly harvested thalli showed an effective quantum yield of PSII [Y(II)] of 0.52 ± 0.06 that decreased to ∼60% of the initial value at 3000 mM sorbitol, and 4000 mM sorbitol led to a complete loss of Y(II). The Y(II) of thalli exposed to controlled desiccating conditions at 60% relative humidity (RH) ceased within 240 min, whereas zero values were reached after 120 min at 20% RH. All investigated samples completely recovered Y(II) within ∼100 min after rehydration. Relative electron transport rates (rETR) were temperature dependent, increasing from 5, 10, to 25 °C but strongly declining at 45 °C. Transmission electron microscopy of samples desiccated for 2.5 h showed an electron dense appearance of the entire cytoplasm when compared to control samples. Thylakoid membranes were still visible in desiccated cells, corroborating the ability to recover. Control and desiccated cells contained numerous storage lipids and starch grains, providing reserves. Overall, P. calophylla showed a high capacity to cope with water-limiting conditions on a physiological and structural basis. A lipophilic outer layer of the cell walls might contribute to reduce water evaporation in this poikilohydric organism.

Preparation of Fe3O4/Reduced Graphene Oxide Nanocomposites with Good Dispersibility for Delivery of Paclitaxel

The Fe3O4/reduced graphene oxide (Fe3O4/RGO) nanocomposites with good dispersibility were synthesized for targeted delivery of paclitaxel (PTX). Firstly, the superparamagnetic Fe3O4/functional GO nanocomposites were prepared via hydrothermal method in which GO sheets were modified by surfactant wrapping. The Fe3O4/RGO nanocomposites were successively prepared through the reduction of graphene oxide. The products were investigated by Fourier-transform infrared spectrum, X-ray diffraction, scanning electron microscopy, transmission electron microscopy, thermogravimetric analysis, and vibration sample magnetometry. It was found that spherical Fe3O4 nanoparticles were uniformly anchored over the RGO matrix and the nanocomposites were superparamagnetic with saturation magnetization (Ms) of 9.39 emu/g. Then PTX was loaded onto Fe3O4/RGO nanocomposites, and the drug loading capacity was 67.9%. Cell viability experiments performed on MCF-7 demonstrated that the Fe3O4/RGO-loaded PTX (Fe3O4/RGO/PTX) showed cytotoxicity to MCF-7, whereas the Fe3O4/RGO displayed no obvious cytotoxicity. The above results indicated that Fe3O4/RGO/PTX nanocomposites had potential application in tumor-targeted chemotherapy.

Transmission Electron Microscopy (TEM) to Study Histology of Pollen and Pollen Tubes.

Here, we describe methods of transmission electron microscopy (TEM) based on conventional chemical fixation and high-pressure freezing (HPF) and freeze-substitution (FS) to examine the ultrastructure of Arabidopsis pollen grains and pollen tubes. Compared to other plant samples, such as root or leaf, pollen grains have thick pollen coat and cell wall which limit the permeation of fixative. Thus, it is difficult to obtain high-quality ultrastructural images of pollen. Moreover, pollen tube is very soft and the traditional procedure is too harsh to get an intact pollen tube sample. Up to now, there is no available mature protocol for TEM sample preparation of Arabidopsis pollen tube. Here, we describe the details and step-by-step procedures of chemical fixation, HPF, FS, and resin-embedding protocols for Arabidopsis pollen and pollen tube. In addition, we also provide a method on how to get longitudinal sections of pollen tubes.

透過電子顕微鏡法の最近の動向とグリーンナノテクノロジーへの応用

Transmission electron microscopy has been playing an important role in research and development of nanotechnology which is common and fundamental in various fields of science and technology such as energy, environmental sciences, information and telecommunications, and life sciences and etc. A transmission electron microscope (TEM) provides us with crystallographic information and positional information from a local area of specimen. Furthermore information on local chemical composition and atomic bonding state is obtained provided the TEM is equipped with an X-ray energy dispersive spectrometer (XEDS) and an electron energy loss spectrometer (EELS). The image resolution and special resolution of elemental analysis have been improved to an atomic level together with the development of an analytical TEM that has a field emission gun, an aberration corrector and a function of scanning TEM (STEM). It has been possible to extract high quality and various information on substance and materials, owing to the development of electron detectors and imaging techniques of STEM. Objects of TEM observation have been widely spread after the development of related techniques such as preparation of TEM samples and controlling of specimen environment (heating, cooling, gas pressure, stress application etc.). We introduce recent trend of transmission electron microscopy and demonstrate some applications to green nanotechnology which is closely related to development of renewable energy, energy saving, energy storing, saving of rear resource, removal of harmful substance from air and water and so on.

How to Get Well-Preserved Samples for Transmission Electron Microscopy

How to Get Well-Preserved Samples for Transmission Electron Microscopy

An electron energy loss spectrometer based streak camera for time resolved TEM measurements

We propose an experimental setup based on a streak camera approach inside an energy filter to measure time resolved properties of materials in the transmission electron microscope (TEM). In order to put in place the streak camera, a beam sweeper was built inside an energy filter. After exciting the TEM sample, the beam is swept across the CCD camera of the filter. We describe different parts of the setup at the example of a magnetic measurement. This setup is capable to acquire time resolved diffraction patterns, electron energy loss spectra (EELS) and images with total streaking times in the range between 100ns and 10μs.

TEM sample preparation by femtosecond laser machining and ion milling for high-rate TEM straining experiments

To model mechanical properties of metals at high strain rates, it is important to visualize and understand their deformation at the nanoscale. Unlike post mortem Transmission Electron Microscopy (TEM), which allows one to analyze defects within samples before or after deformation, in situ TEM is a powerful tool that enables imaging and recording of deformation and the associated defect motion during mechanical loading. Unfortunately, all current in situ TEM mechanical testing techniques are limited to quasi-static strain rates. In this context, we are developing a new test technique that utilizes a rapid straining stage and the Dynamic TEM (DTEM) at the Lawrence Livermore National Laboratory (LLNL). The new straining stage can load samples in tension at strain rates as high as 4×103/s using two piezoelectric actuators operating in bending while the DTEM at LLNL can image in movie mode with a time resolution as short as 70ns. Given the piezoelectric actuators are limited in force, speed, and displacement, we have developed a method for fabricating TEM samples with small cross-sectional areas to increase the applied stresses and short gage lengths to raise the applied strain rates and to limit the areas of deformation. In this paper, we present our effort to fabricate such samples from bulk materials. The new sample preparation procedure combines femtosecond laser machining and ion milling to obtain 300µm wide samples with control of both the size and location of the electron transparent area, as well as the gage cross-section and length.

Microstructure and defect analysis in the vicinity of blisters in polycrystalline tungsten

Up to now, analyzing the production of dislocation-type defects in the subsurface region of plasma or ion-exposed tungsten samples has been hampered by the challenging production of suitable cross-section samples for transmission electron microscopy. We present two reliable methods based on precision electropolishing to prepare cross-sections of tungsten that allow direct imaging of dislocation-type defects by scanning as well as by transmission electron microscopy. Using these methods, we are able to demonstrate a clear enhancement of the dislocation density in the caps of blisters on tungsten exposed to H isotope plasma, i.e., of surface morphologies that are correlated to subsurface cavities. As a benchmark, we also show a cross-section of tungsten irradiated by 20 MeV W6+ ions.

Microstructure Observations of Graphite in Gray Cast Iron Using TEM

Cast iron is an iron alloy mainly composed of carbon and silicon, the amount of carbon is more than 2.1 mass%. Cast irons, gray cast iron and ductile cast iron, have been used as industrial parts and automobile parts widely because they have a good wear resistance and an excellent machinability. Graphite formation mechanism have been proposed, but, it is not established clearly yet. In this study, the microstructure of flake graphite was investigated to reveal the graphite formation mechanisms using FC250 alloy. Transmission electron microscopy (TEM) samples were prepared using focused ion beam (FIB). In the case of a cross section of flake graphite taken perpendicular to its elongated direction using TEM, internal microstructure of flake graphite was observed layered structure. In the case of a cross section of flake graphite taken parallel to its elongated direction, clear microstructure was not observed. Selected area electron diffraction (SAED) from flake graphite showed &lt;0001&gt; direction of graphite are mostly parallel to their thickness.

Novel method for measurement of transistor gate length using energy-filtered transmission electron microscopy

As the feature size of devices continues to decrease, transmission electron microscopy (TEM) is becoming indispensable for measuring the critical dimension (CD) of structures. Semiconductors consist primarily of silicon-based materials such as silicon, silicon dioxide, and silicon nitride, and the electrons transmitted through a plan-view TEM sample provide diverse information about various overlapped silicon-based materials. This information is exceedingly complex, which makes it difficult to clarify the boundary to be measured. Therefore, we propose a simple measurement method using energy-filtered TEM (EF-TEM). A precise and effective measurement condition was obtained by determining the maximum value of the integrated area ratio of the electron energy loss spectrum at the boundary to be measured. This method employs an adjustable slit allowing only electrons with a certain energy range to pass. EF-TEM imaging showed a sharp transition at the boundary when the energy-filter’s passband centre was set at 90 eV, with a slit width of 40 eV. This was the optimum condition for the CD measurement of silicon-based materials involving silicon nitride. Electron energy loss spectroscopy (EELS) and EF-TEM images were used to verify this method, which makes it possible to measure the transistor gate length in a dynamic random access memory manufactured using 35 nm process technology. This method can be adapted to measure the CD of other non-silicon-based materials using the EELS area ratio of the boundary materials.

Liquid-solid phase transition of Ge-Sb-Te alloy observed by in-situ transmission electron microscopy

Melting and crystallization dynamics of the multi-component Ge-Sb-Te alloy have been investigated by in-situ transmission electron microscopy (TEM). Starting point of the phase transition study is an ordered hexagonal Ge1Sb2Te4 thin film on Si(111) where the crystal structure and the chemical composition are verified by scanning TEM and electron energy-loss spectroscopy, respectively. The in-situ observation of the liquid phase at 600°C including the liquid-solid and liquid-vacuum interfaces and their movements was made possible due to an encapsulation of the TEM sample. The solid-liquid interface during melting displays a broad and diffuse transition zone characterized by a vacancy induced disordered state. Although the velocities of interface movements are measured to be in the nanometer per second scale, both, for crystallization and solidification, the underlying dynamic processes are considerably different. Melting reveals linear dependence on time, whereas crystallization exhibits a non-linear time-dependency featuring a superimposed start-stop motion. Our results may provide valuable insight into the atomic mechanisms at interfaces during the liquid-solid phase transition of Ge-Sb-Te alloys.

Gentle transfer method for water- and acid/alkali-sensitive 2D materials for (S)TEM study

We report a method in making transmission electron microscopy sample for both CVD-grown and exfoliated 2D materials without etching process, thus gentle to those 2D materials that are sensitive to water and reactive etchants. Large-scale WS2 monolayer grown on glass, NbS2 atomic layers grown on exfoliated h-BN flakes, and water-sensitive exfoliated TiS2 flakes are given as representative examples. We show that the as-transferred samples not only retain excellent structural integrity down to atomic scale but also have little oxidations, presumably due to the minimum contact with water/etchants. This method paves the way for atomic scale structural and chemical investigations in sensitive 2D materials.

The Application of Paraphenylenediamine Staining for Assessment of Phospholipidosis.

Drug-induced phospholipidosis is characterized by intracellular accumulation of phospholipids with lamellar bodies in cells exposed to xenobiotics. Demonstration of the lamellar bodies by transmission electron microscopy (TEM) is the hallmark for a definitive diagnosis of phospholipidosis. However, the preparation of tissue samples for TEM and their ultrastructural evaluation are technically challenging and time consuming. Paraphenylenediamine (PPD) is essentially a fat stain, and the staining mechanism is based upon the osmication of unsaturated lipids. Thus, the application of PPD staining to osmicated tissue samples is considered an optimal way to identify lipids. We evaluated the potential of PPD staining to localize phospholipid accumulations on osmium-fixed semi-thin tissue sections of the lung, kidney, and liver, which were affected with phospholipidosis, under a light microscope. PPD staining revealed the presence of PPD positive dark fine granular material in the cytoplasm for all affected tissues examined, which correlated ultrastructurally with lamellar bodies as well as a light microscopic finding of cytoplasmic vacuolation. The great advantage of PPD is that it can be incorporated into the protocol for standard TEM tissue preparation and significantly improve the efficiency of TEM work. In conclusion, PPD provides a simple, sensitive, and reliable method for visualizing phospholipid accumulation on light microscopy and represents an easy tool to study drug-induced phospholipidosis.

Fabrication of thin TEM sample of ionic liquid for high-resolution ELNES measurements

Investigation of the local structure, ionic and molecular behavior, and chemical reactions at high spatial resolutions in liquids has become increasingly important. Improvements in these areas help to develop efficient batteries and improve organic syntheses. Transmission electron microscopy (TEM) and scanning-TEM (STEM) have excellent spatial resolution, and the electron energy-loss near edge structure (ELNES) measured by the accompanied electron energy-loss spectroscopy (EELS) is effective to analyze the liquid local structure owing to reflecting the electronic density of states. In this study, we fabricate a liquid-layer-only sample with thickness of single to tens nanometers using an ionic liquid. Because the liquid film has a thickness much less than the inelastic mean free path (IMFP) of the electron beam, the fine structure of the C-K edge electron energy loss near edge structure (ELNES) can be measured with sufficient resolution to allow meaningful analysis. The ELNES spectrum from the thin liquid film has been interpreted using first principles ELNES calculations.

Small versus Large Iron Oxide Magnetic Nanoparticles: Hyperthermia and Cell Uptake Properties.

Efficient use of magnetic hyperthermia in clinical cancer treatment requires biocompatible magnetic nanoparticles (MNPs), with improved heating capabilities. Small (~34 nm) and large (~270 nm) Fe3O4-MNPs were synthesized by means of a polyol method in polyethylene-glycol (PEG) and ethylene-glycol (EG), respectively. They were systematically investigated by means of X-ray diffraction, transmission electron microscopy and vibration sample magnetometry. Hyperthermia measurements showed that Specific Absorption Rate (SAR) dependence on the external alternating magnetic field amplitude (up to 65 kA/m, 355 kHz) presented a sigmoidal shape, with remarkable SAR saturation values of ~1400 W/gMNP for the small monocrystalline MNPs and only 400 W/gMNP for the large polycrystalline MNPs, in water. SAR values were slightly reduced in cell culture media, but decreased one order of magnitude in highly viscous PEG1000. Toxicity assays performed on four cell lines revealed almost no toxicity for the small MNPs and a very small level of toxicity for the large MNPs, up to a concentration of 0.2 mg/mL. Cellular uptake experiments revealed that both MNPs penetrated the cells through endocytosis, in a time dependent manner and escaped the endosomes with a faster kinetics for large MNPs. Biodegradation of large MNPs inside cells involved an all-or-nothing mechanism.