Quantification of extreme thermal gradients during in situ transmission electron microscope heating experiments

Studies on materials affected by large thermal gradients and rapid thermal cycling are an area of increasing interest, driving the need for real time observations of microstructural evoultion under transient thermal conditions. However, current in situ transmission electron microscope (TEM) heating stages introduce uniform temperature distributions across the material during heating experiments. Here, a methodology is described to generate thermal gradients across a TEM specimen by modifying a commercially available MEMS-based heating stage. It was found that a specimen placed next to the metallic heater, over a window, cut by FIB milling, does not disrupt the overall thermal stability of the device. Infrared thermal imaging (IRTI) experiments were performed on unmodified and modified heating devices, to measure thermal gradients across the device. The mean temperature measured within the central viewing area of the unmodified device was 3–5% lower than the setpoint temperature. Using IRTI data, at setpoint temperatures ranging from 900 to 1,300°C, thermal gradients at the edge of the modified window were calculated to be in the range of 0.6 × 10⁶ to 7.0 × 10⁶°C/m. Additionally, the Ag nanocube sublimation approach was used, to measure the local temperature across a FIB-cut Si lamella at high spatial resolution inside the TEM, and demonstrate “proof of concept” of the modified MEMS device. The thermal gradient across the Si lamella, measured using the latter approach was found to be 6.3 × 10⁶°C/m, at a setpoint temperature of 1,000°C. Finally, the applicability of this approach and choice of experimental parameters are critically discussed.     

Direct observation of liquid crystals using cryo‐TEM: Specimen preparation and low‐dose imaging

Liquid crystals (LCs) represent a challenging group of materials for direct transmission electron microscopy (TEM) studies due to the complications in specimen preparation and the severe radiation damage. In this paper, we summarize a series of specimen preparation methods, including thin film and cryo‐sectioning approaches, as a comprehensive toolset enabling high‐resolution direct cryo‐TEM observation of a broad range of LCs. We also present comparative analysis using cryo‐TEM and replica freeze‐fracture TEM on both thermotropic and lyotropic LCs. In addition to the revisits of previous practices, some new concepts are introduced, e.g., suspended thermotropic LC thin films, combined high‐pressure freezing and cryo‐sectioning of lyotropic LCs, and the complementary applications of direct TEM and indirect replica TEM techniques. The significance of subnanometer resolution cryo‐TEM observation is demonstrated in a few important issues in LC studies, including providing direct evidences for the existence of nanoscale smectic domains in nematic bent‐core thermotropic LCs, comprehensive understanding of the twist‐bend nematic phase, and probing the packing of columnar aggregates in lyotropic chromonic LCs. Direct TEM observation opens ways to a variety of TEM techniques, suggesting that TEM (replica, cryo, and in situ techniques), in general, may be a promising part of the solution to the lack of effective structural probe at the molecular scale in LC studies. Microsc. Res. Tech. 77:754–772, 2014. © 2014 Wiley Periodicals, Inc.     

User‐independent EBSD parameters to study the progress of recovery and recrystallization in Cu–Zn alloy during in situ heating

Microstructural evolution of cold‐rolled Cu–5%Zn alloy during in situ heating inside field‐emission scanning electron microscope was utilized to obtain user‐independent parameters in order to trace the progress of static recovery and recrystallization. Electron back‐scattered diffraction (EBSD)‐based orientation imaging microscopy was used to obtain micrographs at various stages of in situ heating. It is shown that unlike the pre‐existing methods, additional EBSD‐based parameter can be used to trace the progress of recovery and recrystallization, which is not dependent on user input and hence less prone to error. True strain of 0.3 was imposed during cold rolling of alloy sample. Rolled sample was subjected to in situ heating from room temperature to 500°C (～0.58 Tm) with soaking time of 10 min, at each of the intermediate temperatures viz. 100, 200, 300, 400 and 450°C. After reaching 500°C, the sample was kept at this temperature for a maximum duration of around 15 h. The sample showed clear signs of recovery for temperature up to 450°C, and at 500°C, recrystallization started to take place. Recrystallization kinetics was moderate, and full recrystallization was achieved in approximately 120 min. We found that EBSD parameter, namely, band contrast intensity can be used as an extra handle to map out the progress of recrystallization occurring in the sample. By contrast, mean angular deviation can be used to understand the evolution of recovery in samples. The parameters mentioned in the current study, unlike other pre‐existing methods, can also be used for mapping local microstructural transformations due to recovery and recrystallization. We discuss the benefits and limitations in using these additional handles in understanding the changes taking place in the material during in situ heating.     

Making the practically impossible “Merely difficult”—Cryogenic FIB lift‐out for “Damage free” soft matter imaging

The preparation of thinned lamellae from bulk samples for transmission electron microscopy (TEM) analysis has been possible in the focussed ion beam scanning electron microscope (FIB‐SEM) for over 20 years via the in situ lift‐out method. Lift‐out offers a fast and site specific preparation method for TEM analysis, typically in the field of materials science. More recently it has been applied to a low‐water content biological sample (Rubino 2012). This work presents the successful lift‐out of high‐water content lamellae, under cryogenic conditions (cryo‐FIB lift‐out) and using a nanomanipulator retaining its full range of motion, which are advances on the work previously done by Rubino (2012). Strategies are explored for maintaining cryogenic conditions, grid attachment using cryo‐condensation of water and protection of the lamella when transferring to the TEM. Microsc. Res. Tech. 79:298–303, 2016. © 2016 Wiley Periodicals, Inc.     

An improved preparation method for cross‐sectional TEM specimens of films deposited on metallic substrates

Cross‐sectional TEM analysis is one of the most important techniques to characterize microstructures of films. However, the complex process, low efficiency, and low success rate of specimen preparation limit its application. This paper analyzed the main causes of low success rate and proposed an improved method for specimen preparation of films deposited on metallic substrates. This method consisting of twin‐jet electropolishing and one‐sided rocking ion milling is high in efficiency and success rate. Microsc. Res. Tech. 79:276–279, 2016. © 2016 Wiley Periodicals, Inc.     

In situ TEM studies of micron‐sized all‐solid‐state fluoride ion batteries: Preparation,prospects, and challenges

Trustworthy preparation and contacting of micron‐sized batteries is an essential task to enable reliable in situ TEM studies during electrochemical biasing. Some of the challenges and solutions for the preparation of all‐solid‐state batteries for in situ TEM electrochemical studies are discussed using an optimized focused ion beam (FIB) approach. In particular redeposition, resistivity, porosity of the electrodes/electrolyte and leakage current are addressed. Overcoming these challenges, an all‐solid‐state fluoride ion battery has been prepared as a model system for in situ TEM electrochemical biasing studies and first results on a Bi/La_0.9Ba_0.1F_2.9 half‐cell are presented. Microsc. Res. Tech. 79:615–624, 2016. © 2016 Wiley Periodicals, Inc.     

Extraction of structural and chemical information from high angle annular dark‐field image by an improved peaks finding method

With the development of spherical aberration (Cs) corrected scanning transmission electron microscopy (STEM), high angle annular dark filed (HAADF) imaging technique has been widely applied in the microstructure characterization of various advanced materials with atomic resolution. However, current qualitative interpretation of the HAADF image is not enough to extract all the useful information. Here a modified peaks finding method was proposed to quantify the HAADF‐STEM image to extract structural and chemical information. Firstly, an automatic segmentation technique including numerical filters and watershed algorithm was used to define the sub‐areas for each atomic column. Then a 2D Gaussian fitting was carried out to determine the atomic column positions precisely, which provides the geometric information at the unit‐cell scale. Furthermore, a self‐adaptive integration based on the column position and the covariance of statistical Gaussian distribution were performed. The integrated intensities show very high sensitivity on the mean atomic number with improved signal‐to‐noise (S/N) ratio. Consequently, the polarization map and strain distributions were rebuilt from a HAADF‐STEM image of the rhombohedral and tetragonal BiFeO₃ interface and a MnO₂ monolayer in LaAlO₃/SrMnO₃/SrTiO₃ heterostructure was discerned from its neighbor TiO₂ layers. Microsc. Res. Tech. 79:820–826, 2016. © 2016 Wiley Periodicals, Inc.     

A hot stage SEM for gas-solid reaction studies

An ultra-high vacuum, field emission source, scanning electron microscope (SEM) equipped with a gas cell to permit the in situ study of high temperature gas-solid reactions is described. High resolution SEM (50 Å) and scanning Auger microscopy (300 Å resolution) can be carried out at specimen temperatures up to 1000°C. Reaction products and processes can be imaged continuously in the SEM at pressures up to ～0.2 Torr. Gas pressures up to 50 Torr are possible but with some limitations. Methods of specimen mounting, problems of temperature measurement, and the microscope performance at high specimen temperatures and gas pressures are discussed. Examples of metal surface preparation and impurity segregation under high temperature annealing and gas exposure are reported.     

High‐resolution cryo‐SEM allows direct identification of F‐actin at the inner nuclear membrane of Xenopus oocytes by virtue of its structural features

The nuclear envelope of Xenopus laevis stage VI oocytes was studied in a high‐resolution field emission cryo‐scanning electron microscope to compare the level of structural preservation obtainable by different procedures of specimen preparation. All approaches generally allowed frequent detection of long filaments of about 10 nm in diameter that were attached to the nuclear envelope's inner membrane facing the nuclear interior. Structural details of these 10‐nm filaments, however, could not be unveiled by standard procedures of specimen preparation and analysis, including critical point drying and imaging at room temperature. In contrast, after freeze‐drying and imaging at ?100°C, the 10‐nm filament type was found to be composed of distinct globular subunits of approximately 5 nm in diameter that were arranged in a helical manner with right‐handed periodicity. Stereoscopic images showed that some of these filaments were lying directly on the membrane whereas others appeared to hover at a certain distance above the nuclear envelope. The appearance of these filaments was highly similar to that of in vitro polymerized F‐actin analysed in parallel, and closely resembled the structural characteristics of F‐actin filaments described earlier. By virtue of their structural features we therefore conclude that these filaments at the nuclear periphery represent F‐actin. The high level of structural resolution obtainable by field emission cryo‐SEM illustrates the potential of this method for studying details of biological structures in a subcellular context.     

Two‐In‐one sample preparation for plan‐VIew TEM

Transmission electron microscopy (TEM) sample preparation requires special skills, it is time consuming and costly, hence, an increase of the efficiency is of primary importance. This article describes a method that duplicates the yield of the conventional mechanical and ion beam preparation of plan‐view TEM samples. As a modification of the usual procedures, instead of one two different samples are comprised in a single specimen. The two pre‐cut slabs, one from each samples, are embedded side by side in the window of a 3 mm dia Ti disk and the specimen is thinned mechanically and by ion milling until perforation that occurs at the interface of the two different slabs. That, with proper implementation, provides acceptable size thin area for the TEM study of both samples. The suitability of the two‐in‐one method has been confirmed through examples. Microsc. Res. Tech. 78:599–602, 2015. © 2015 Wiley Periodicals, Inc.     

Development of high‐temperature strain instrumentation for in situ SEM evaluation of ductility dip cracking

Nowadays, the implementation of sophisticated in situ electron microscopy tests is providing new insights in several areas. In this work, an in situ high‐temperature strain test into a scanning electron microscope was developed. This setup was used to study the grain boundary sliding mechanism and its effect on the ductility dip cracking. This methodology was applied to study the mechanical behaviour of Ni‐base filler metal alloys ERNiCrFe‐7 and ERNiCr‐3, which were evaluated between 700°C and 1000°C. The ductility dip cracking susceptibility (threshold strain; ε_min) for both alloys was quantified. The ε_min of ERNiCrFe‐7 and ERNiCr‐3 alloys were 7.5% and 16.5%, respectively, confirming a better resistance of ERNiCr‐3 to ductility dip cracking. Furthermore, two separate components of grain boundary sliding, pure sliding (S_p) and deformation sliding (S_d), were identified and quantified. A direct and quantitative link between grain boundary tortuosity, grain boundary sliding and ductility dip cracking resistance has been established for the ERNiCrFe‐7 and ERNiCr‐3 alloys.     

Alkyloxy‐s‐triazine derivatives with and without fluorine‐containing substituents as lubricants for steel–steel contact

Three alkyloxy‐s‐triazine derivatives were synthesized and their tribological properties as lubricants for steel–steel contact were evaluated using an Optimol SRV tester at 20°C and 100°C. Their thermal stabilities were also investigated by thermogravimetric analysis. The results show that the three alkyloxy‐s‐triazine lubricants have good thermal stability. Moreover, 2,4,6‐tris(1,1,5‐tri‐H‐octafluoropentyloxy)‐1,3,5‐s‐triazine (FPOT) possesses the best anti‐wear property and good load‐carrying capacity both at 20°C and 100°C. At 20°C the anti‐wear effectiveness of 2,4,6‐tris(n‐pentyloxy)‐1,3,5‐s‐triazine (POT) is the worst, while at 100°C that of the 1,1,5‐tri‐H‐octafluoropentyloxy and/or 1,1,7‐tri‐H‐dodecafluoroheptyloxy tri‐substituted s‐triazine mixture (FMOT) is the worst. In addition, the friction‐reducing properties of the two fluoroalkyloxy‐s‐triazines, FPOT and FMOT, are not as good as those of the non‐fluorine‐containing alkyloxy‐s‐triazine POT. Scanning electron spectroscopy with an energy dispersive analyzer of X‐ray and X‐ray photoelectron spectroscopy analyses of the worn surface indicate that during the rubbing process, tribochemical reactions occur between the lubricants and the metal surface to generate a complex boundary lubrication film comprised of FeF₂, Fe(OH)₂, organofluorine and organonitrogen compounds. Copyright © 2006 John Wiley & Sons, Ltd.     

Visualization of the native shape of bodipy‐labeled DNA in Escherichia coli by correlative microscopy

The native shape and intracellular distribution of newly synthesized DNA was visualized by correlative (light and electron) microscopy in ice embedded whole cells of Escherichia coli. For that purpose, the commercially available modified nucleoside triphosphate named BODIPY® FL‐14‐dUTP was enzymatically incorporated in vivo into the genome of E. coli mutant K12 strain, which cannot synthesize thymine. The successful incorporation of this thymidine analogue was confirmed first by fluorescence microscope, where the cells were stained in the typical for bodipy green color. Later the preselected labeled E. coli were observed by Hilbert Differential Transmission Electron Microscope (HDC TEM) and the distribution of elemental boron (contained in bodipy) was visualized at high‐resolution by an electron spectroscopic imaging (ESI) technique. The practical detection limit of boron was found to be around 5 ～ 10 mmol/kg in area of 0.1 μm², which demonstrated that ESI is a suitable approach to study the cytochemistry and location of labeled nucleic fragments within the cytoplasmic chromosomal area. In addition, the fine cellular fibrous and chromosomal ultrastructures were revealed in situ by combing of phase‐plate HDC TEM and ESI. The obtained results conclude that the correlation between fluorescent microscopy with phase‐plate HDC TEM and ESI is a powerful approach to explore the structural and conformation dynamics of DNA replication machinery in frozen cells close to the living state.     

Off-axis electron holography of electrostatic potentials in unbiased and reverse biased focused ion beam milled semiconductor devices

Off‐axis electron holography in the transmission electron microscope (TEM) is used to measure two‐dimensional electrostatic potentials in both unbiased and reverse biased silicon specimens that each contain a single p–n junction. All the specimens are prepared for examination in the TEM using focused ion beam (FIB) milling. The in situ electrical biasing experiments make use of a novel specimen geometry, which is based on a combination of cleaving and FIB milling. The design and construction of an electrical biasing holder are described, and the effects of TEM specimen preparation on the electrostatic potential in the specimen, as well as on fringing fields beyond the specimen surface, are assessed.     

Laser‐induced reversion of precipitates in an Al‐Li alloy: Study on temperature rise in pulsed laser atom probe

The influence of tuning the laser pulse energy during the analyses on the resulting microstructure in a specimen utilizing an ultra‐fast laser assisted atom probe was demonstrated by a case study of a binary Al‐Li alloy. The decomposition parameters, such as the size, number density, volume fraction, and composition of precipitates, were carefully monitored after each analysis. A simple model was employed to estimate the corresponding specimen temperature for each value of the laser energy. The results indicated that the corresponding temperatures for the laser pulse energy in the range of 10 to 80 pJ are located inside the miscibility gap of the binary Al‐Li phase diagram and fall into the metastable equilibrium field. In addition, the corresponding temperature for a laser pulse energy of 100 pJ was in fairly good agreement with reported range of solvus temperature, suggesting a result of reversion upon heating due to laser pulsing. Microsc. Res. Tech. 79:727–737, 2016. © 2016 Wiley Periodicals, Inc.     

A comparative study on the self‐assembly of an amyloid‐like peptide at water–solid interfaces and in bulk solutions

In the past years the self‐assembly of amyloid‐like peptides has attracted increasing attentions, because it is highly related to neurodegenerative diseases and has a potential for serving as nanomaterial to fabricate novel and useful nanostructures. In this paper, we focused on the role of interfacial conditions in the self‐assembly of an amyloid‐like peptide, termed Pep11. It was found that, when dissolved in bulk solutions, Pep11 formed into β‐sheet structures and assembled into long filaments in several hours, as revealed by Thioflavin T fluorescence and transmission electron microscopy (TEM) morphology characterization, respectively. When the peptide solution was added onto a mica/HOPG substrate, peptide filaments with three preferred orientations with an angle of 60° to each other were formed immediately, as imaged in situ by atomic force microscopy (AFM). However, the kinetics in filament formation and the morphologies of the formed beta sheet either on HOPG and mica or in bulk solutions were quite different. These results indicate that the interfacial properties dramatically affect the peptide self‐assembly process. Microsc. Res. Tech. 78:375–381, 2015. © 2015 Wiley Periodicals, Inc.     

EBSD coupled to SEM in situ annealing for assessing recrystallization and grain growth mechanisms in pure tantalum

An in situ annealing stage has been developed in‐house and integrated in the chamber of a Scanning Electron Microscope equipped with an Electron BackScattered Diffraction system. Based on the Joule effect, this device can reach the temperature of 1200°C at heating rates up to 100°C/s, avoiding microstructural evolutions during heating. A high‐purity tantalum deformed sample has been annealed at variable temperature in the range 750°C–1030°C, and classical mechanisms of microstructural evolutions such as recrystallization and grain coarsening phenomena have been observed. Quantitative measurements of grain growth rates provide an estimate of the mean grain boundary mobility, which is consistent with the value estimated from physical parameters reported for that material. In situ annealing therefore appears to be suited for complementing bulk measurements at relatively high temperatures, in the context of recrystallization and grain growth in such a single‐phase material.     

The effect of an Ni–Cr protective layer on cyclic oxidation of Ti3Al

The effect of an 80Ni?20Cr (at.%) metallic coating on the cyclic oxidation behaviour of a Ti₃Al‐based alloy with the composition Ti?25Al?11Nb (at.%) was investigated in this study. Cyclic oxidation tests were carried out in air at 600 °C and 900 °C for 120 h. For one cycle test, the specimens were held for 24 h at test temperature and then furnace‐cooled to room temperature. The oxidation rate was determined by plotting the mass gain per unit surface area of the specimen vs. exposure time. The morphology and composition of the oxidation products were characterized on the cross‐section of the specimens by scanning electron microscopy, energy‐dispersive X‐ray spectroscopy and atomic force microscopy. The oxidation scale forms during exposure at both 600 °C and 900 °C. TiO₂ is the main oxide component, whereas the Al₂O₃ layer appears only discontinuously. The remarkable improvement in oxidation resistance at 900 °C was attributed to the chemical composition and structure of the scale formed on the 80Ni?20Cr coating.     

Particle size analysis: 90Y and 99mTc‐labelled colloids

Colloidal particle size is an important characteristic to consider when choosing a radiopharmaceutical for diagnosis and therapeutic purposes in nuclear medicine. Photon correlation spectroscopy (PCS) and transmission electron microscopy (TEM) were used to determine the particle‐size distribution of ⁹⁰Y‐ and ^99mTc‐labelled antimony trisulfide (Sb₂S₃) and tin colloids (Sn‐colloid). ⁹⁰Y‐Sb₂S₃ and ^99mTc‐Sb₂S₃ were found to have a diameter of 28.92 ± 0.14 and 35.61 ± 0.11 nm, respectively, by PCS. By TEM, ⁹⁰Y‐Sb₂S₃ particles were measured to be 14.33 ± 0.09 nm. ⁹⁰Y‐labelled Sn colloid were found to exist with a d_v(max1) of 805 nm and a d_v(max2) of 2590 nm, by PCS, whereas ^99mTc‐Sn colloid was shown to have more than 80% of radioactive particles of approximately 910 nm by PCS. For ⁹⁰Y‐labelled Sb₂S₃ and Sn colloid, a comparison of TEM and PCS indicates that these techniques found significantly different mean diameters. TEM has an excellent resolution necessary for radiocolloid particle‐sizing analysis, and it is a desirable size‐measuring technique because it is more reliable than PCS.     

Cyclic fatigue resistance and energy dispersive X‐ray spectroscopy analysis of novel heat‐treated nickel–titanium instruments at body temperature

The aim of this study was to evaluate the cyclic fatigue resistance of Dia‐X, WaveOne Gold and One Curve files in a water bath at intracanal temperature (35°C). Thirty‐nine instruments Dia‐X, WaveOne Gold, and One Curve systems (n = 13) were tested in an artificial canal with a curvature angle of 60° and a radius of 3 mm. A water bath setup at a temperature of 35°C was used to simulate the intracanal temperature and time to fracture (TTF) as seconds was recorded. The mean data were analyzed statistically using one‐way ANOVA, and post hoc Tukey test (p = .05). The fractured surface of the instruments was examined with scanning electron microscopy (SEM) and chemical composition of the instruments were investigated with energy dispersive X‐ray spectroscopy (EDS). Statistically significant differences were detected in TTF values of all the systems as follows: One Curve > WaveOne Gold > Dia‐X (p < .05). One Curve instruments demonstrated the highest TTF values in all the tested instruments. The EDS microanalysis revealed similar NiTi composition of on the surface of One Curve, WaveOne Gold, and Dia‐X instruments. The novel manufacturing process, including C‐wire heat treatment and the variable cross‐section of the One Curve files, could be the main factors affecting the fatigue life of the instruments.