Microscope Chamber Research Articles

Local strain heterogeneity and damage mechanisms in zirconia particle-reinforced TRIP steel MMCs: in situ tensile testing with digital image processing

In this work, the microstructural deformation and damage mechanisms of TRIP steel metal matrix composites (MMCs) reinforced with Magnesia Partially Stabilized Zirconia (Mg-PSZ) particles are investigated by employing in situ tensile testing within a scanning electron microscope chamber, complemented by digital image correlation and advanced image processing techniques. The study is carried out on samples with varied volume fractions (0%, 10%, and 20%) of zirconia particles and damage mechanisms in different samples under specified loading conditions. Through both qualitative and quantitative assessments of deformation, damage, and clustering, the investigation provides a comprehensive understanding of the distribution and damage initiation. The study findings reveal that, generally, the steel matrix exhibits high toughness, with minimal occurrences of microcracking at high strains that cause significant damage. In samples with increasing particle content, delamination at the matrix–particle interface and cracking of Mg-PSZ particles were found to be critical contributors to material failure and were quantitatively analyzed using computational analyses conducted with MATLAB. The work highlights the initiation and evolution of each damage mechanism in zirconia particle-reinforced TRIP steel MMCs to facilitate scientists and engineers in improving manufacturing and application decisions in industries such as automotive, aerospace, and heavy machinery, which demand materials with exceptional toughness and durability.Graphical

The influence of the hydrogenation process on the microstructure and properties of metallic materials

Hydrogen, which belongs to the group of the smallest elements, can freely penetrate the structure of metallic materials and cause enormous damage. This phenomenon may occur due to various conditions and may have different effects on the properties of materials. Understanding these mechanisms is important in the era of changing to alternative energy sources. This situation may occur in combustion engines powered by hydrogen-enriched fuel. The work analyzed the impact of various hydrogenation processes on the properties of aesthetic steel, which is allergic to the formation of carbides at the grain boundaries, such a microstructure is intended to simulate the least favorable working conditions. The work was aimed at evaluating the effects of different hydrogenation methods on changes in the properties of the AISI 310s steel membrane. In the work, electrolytic hydrogenation and heat treatment procedures in a hydrogen atmosphere were used. Investigations of changes in material properties included microhardness and in-situ strength tests in a scanning electron microscope chamber. Research has shown that both processes increase the hardness of materials and change the characteristics of stretching curves.

Advanced ice lithography system using a micro-machined Joule-Thomson cooler

This study introduces a novel ice lithography system integrated with a low-vibration micro-machined Joule-Thomson cooler. Ice lithography is an eco-friendly method for high-resolution nanofabrication on delicate substrates and requires to operate below 130 K in the vacuum of a microscope chamber. Previously, liquid nitrogen rather than cryocoolers was used to cool the system due to its low vibration, but it is both bulky and costly. To overcome these challenges, this study employs a low-vibration micro-machined cooler in a scanning electron microscope (SEM) for ice lithography. The design and methodology of the system are described in detail. The results show that the substrate can reach a temperature of 105 K within 30 minutes with a measured mechanical vibration of less than 10 nm, enabling high-resolution nanofabrication. As proof of concept, the system successfully fabricates nanoscale patterns on a silicon wafer. This low-vibration cooling system has great potential for use in cryogenic electron microscopes (cryo-EMs) in the future.

Spatiotemporal prediction and characterization of microstructure evolution during in-situ heat treatment using a deep learning algorithm

The austenite grain growth plays a significant role in improving the structures and properties of carbon steels. Modeling, characterizing, and predicting the temporal evolution of microstructures is crucial for understanding the relationship of processing-structure-property. Therefore, in this study, we designed a customized in-situ heating device that could be installed in the scanning electron microscope chamber to observe the temporal evolution process of the microstructures of 45# steel. We collected the in-situ experimental images during heating for downstream deep learning-based grain boundary extraction, spatiotemporal sequence characterization, and prediction. The proposed pipeline was fully verified in three experimental datasets under two distinct heating schemes in terms of (1) short-term and long-term prediction, (2) feeding different lengths of input sequence, and (3) transferring to other datasets. The proposed pipeline presented a high performance in quantitative evaluation metrics and qualitative comparisons between the predicted results and the ground truths in all three verification scenarios. These results are inspiring for researchers as they can preview the evolutional state in advance, thereby saving money, labor, and time with the assistance of deep learning-based predictive models. We believe that this pipeline opens opportunities for modeling, characterizing, and predicting evolutional state, which will aid in the analysis of processing-structure-property relationship, tailoring heat treatment, and improving material properties.

Fracture Toughness Characteristics of Stacked Bilayer Graphene via Far-Field Displacement Measurements.

Mechanical properties of graphene, e.g., strength, modulus, and fracture toughness are extremely sensitive to flaws. Here the fracture properties of stacked bilayer graphene sheets (SBLG) are reported, obtained via stacking two individually grown graphene sheets. The SBLG is presented here as a building block for flaw-resilient nanomaterials. The fracture properties of freestanding SBLG sheets, suspended on transmission electron microscope (TEM) grids, are characterized by stretching the TEM grid inside an scanning electron microscope (SEM) chamber and monitoring the local displacements in real-time. The fracture toughness is measured and expressed as a function of the critical displacement required to propagate existing cracks in the experiment via computational models. This approach decouples force and displacements measurements, and utilizes the known elastic modulus along with the known displacement boundary conditions at the onset of crack growth to estimate the far field force and stress. This strategy represents a breakthrough in nanoscale fracture mechanics for statistical analysis and high throughput experimens on multiple samples at a time. Results demonstrate that the SBLG is markedly tougher than as-grown single or multilayer graphene, with a mode I fracture toughness of ≈28.06±7.5 MPa . The mechanisms leading to a higher toughness of SBLG are also analyzed and discussed.

Hybridoma Screening by Antibody Capture: Detection of Cell-Surface Binding by Immunofluorescence.

If the antigen of interest is a cell-surface protein, immunofluorescence can be used to identify hybridomas secreting monoclonal antibodies to these proteins. They can be stained on microscope chamber slides, flat-bottomed cell culture plates (96, 48, or 24 well), or imaging plates (96 well). This protocol uses 96-well imaging plates.

Line Tension and Drop Size Dependence of Contact Angle at the Nanoscale

Despite considerable research efforts, the influence of contact line tension during wetting at the nanoscale and its experimental determination remain challenging tasks. So far, molecular dynamics simulations and atomic force microscope measurements have contributed to the understanding of these phenomena. However, a direct measurement of the size dependence of the contact angle and the magnitude of the apparent line tension has not been realized so far. Here, we show that the contact angle is indeed dependent on the drop size for small drop diameters and determine the magnitude of the apparent line tension via liquid-metal based measurements of advancing and receding contact angle inside a scanning electron microscope. For this purpose, a robotic setup inside an electron microscope chamber and oxide-free Galinstan droplets—produced via an electromigration-based and focused ion beam irradiation-assisted process—are employed. Using the first-order correction of Young’s equation, we find an apparent line tension value of 4.02 × 10−7 J/m for Galinstan© on stainless steel.

Development of a novel testing methodology for in-situ microstructural characterisation during continuous strain path change

Strain path transition is a common phenomenon during continuous stamping operations of sheet metal and can potentially alter the forming limit of the material. Enabling strain path change in a single experiment is a key challenge faced by several researchers. To understand the effect of continuous strain path change on material forming limits, detailed material characterisation is needed where the material is deformed while the strain path of the material is changed continuously. In this work, a test method was developed, which consisted of a novel mechanical rig and specimen design. The mechanism allowed DP600 steel and AA5182-O aluminium samples to change strain paths continuously without unloading the specimen. Digital image correlation measurements of the strain evolution of the samples during tests showed that the technique was able to alter the strain path of the sample from uniaxial to biaxial strain path. In particular, the measurements showed that the transition from uniaxial to biaxial strain paths was sharper in DP600 than AA5182-O. The test was repeated in a scanning electron microscope (SEM) chamber to observe the behaviour of the microstructure during the strain path transition. The microstructural strain evolution showed rotation of strain bands while the evolution of electron back scattered diffraction (EBSD) maps conveyed grain rotation during continuous strain path change in both the materials. The strain path transition controlled the rotation of grains with preferred crystallographic orientations.

In situ ESEM using 3-D printed and adapted accessories to observe living plantlets and their interaction with enzyme and fungus

This paper describes an innovative way of using environmental scanning electron microscopy (ESEM) and the development of a suitable accessory to perform in situ observation of living seedlings in the ESEM. We provide details on fabrication of an accessory that proved to be essential for such experiments but inexpensive and easy to build in the laboratory, and present our in situ observations of the tissue and cell surfaces. Sample-specific configurations and optimized tuning of the ESEM were defined to maintain Arabidopsis and flax seedlings viable throughout repetitive exposure to the imaging conditions in the microscope chamber. This method permitted us to identify cells and tissues of the live plantlets and characterize their surface morphology during their early stage of growth and development. We could extend the application of this technique, to visualize the response of living cells and tissues to exogenous enzymatic treatments with polygalacturonase in Arabidopsis, and their interaction with hyphae of the wilt fungus Verticillium dahliae during artificial infection in flax plantlets. Our results provide an incentive to the use of the ESEM for in situ studies in plant science and a guide for researchers to optimize their electron microscopy observation in the relevant fields.

In-situ characterization on the fracture behavior of three dimensional polymer nanocomposites reinforced by CNT sponge

This paper reported the fracture behavior of polymer-based nanocomposites reinforced by carbon nanotubes (CNTs). In order to solve the problem associated with the dispersion and re-agglomeration of CNTs, three dimensional CNT sponge was prepared and incorporated into the epoxy through the monomer infusion process. The fracture toughness and critical energy release rate of the nanocomposites were enhanced by more than 100% and 250%, respectively. The toughening mechanism behind such observation was revealed by studying the in-situ crack initiation and propagation in the bulky nanocomposites with assistance of micro-mechanical testing device in a scanning electronic microscope chamber. The results showed that the intrinsic fracture behaviors, such as void generation, as well as the extrinsic toughening features arising from crack deflection and bifurcation, were responsible for the improved fracture properties of nanocomposites.

Microscale solute flow probed with rotating microbead trapped in optical vortex

The dynamics of solute flow in the microscopic chamber can be studied with optical tweezers. A method based on the metallic microbeads trapped in the focused optical vortex beam is proposed. This annular beam of a twisted wavefront exerts torque on a reflective object placed inside the dark core of the vortex. The induced rotational movement of the bead is sensitive to local viscosity changes in the surrounding medium, for example, during the ongoing dissolution process. Two experimental configurations are described, both relying on tracing the angular velocity of the bead in time. In one-bead configuration, the dynamics of local solute concentration can be studied. In two-bead case, the direction and speed of solute flow can be probed with a spatial resolution of single micrometers. We approach the elementary problem of sucrose dissolution and diffusion in water. The surprising impression of the reverse solute flow was observed. Further experimental investigation led to the discovery that this phenomenon originates from the sucrose stream-like diffusion in the mid-depth of the measurement chamber. The rotating microbead method applies for various solid and liquid substances and may become a useful technique for microfluidics research.Graphic abstract

Chemical Gardens Under Mars Conditions: Imaging Chemical Garden Growth In Situ in an Environmental Scanning Electron Microscope

AbstractWe have performed and visualized chemical garden growth experiments directly in situ in an environmental scanning electron microscope. The microscope chamber simulates the surface of Mars. We demonstrate that chemical gardens can form under the conditions of temperature and pressure existing on Mars in the presence of liquid water, silicates, and metal salts. Using chemical gardens from aluminum salts, tubes were formed where the external surface is formed principally by silicate and the inner surface is predominantly aluminum oxide‐hydroxide. It should thus be expected that similar growths of metal salts may be found in geological explorations of water‐rich environments during the search for life on Mars.

In-situ study of strain and texture evolution during continuous strain path change

Automotive stamping is a multi-stage process where a sheet material is drawn in first stage and then redrawn, flanged and pierced in subsequent stages. In the first draw stage, continuous strain path change is induced in the material while a discontinuous strain path change occurs when the material is processed in the subsequent stages of a multi-stage stamping operation. The strain path transition can potentially alter the forming limit of the material. Previous research has investigated the effect of the discontinuous mode of strain path change by loading the sample in one strain path, unloading it, then reloading it in a second path. Thus, discontinuous strain path change was obtained. In this work, the effect of continuous strain path change was investigated with a novel experimental design that allowed cruciform samples to change strain path continuously without unloading. The work was carried out in two stages. In the first stage, the design of the cruciform sample was verified with finite element modelling to ensure the occurrence of continuous strain path change and this was validated experimentally using DX54 material by capturing full-field strain measurements data using digital image correlation technique. The size of the experimental apparatus permitted it to be placed inside a scanning electron microscope chamber. In the second stage, the validated test method was used to evaluate microstructural changes during the deformation including full-field strain and texture evolution. The micro-strain evolution showed rotation of strain bands while the texture evolution conveyed grain rotation during continuous strain path change.

Kinetic models applied to the growth of Saccharomyces boulardii

Predictive microbiology is an interesting tool that allows evaluating the behavior of biomass and metabolites in different culture media, providing multiple benefits, whether scientific or industrial, for these and other reasons the objective of this research was to evaluate applied sigmoidal kinetic models to the growth of Saccharomyces boulardii in milk. For this purpose, flasks were prepared with 200 mL of fresh cow's milk, previously sterilized at 121 ° C x 15 min, then the strains were inoculated at a temperature of 37 ° C and incubated at the same temperature under constant shaking of 20 revolutions per minute (rpm) in a water bath with shaking, for 7 h; To construct the curves and obtain the growth constants, colony-forming units were counted per milliliter (cfu / ml) at one-hour intervals, with a monocular microscope and Neubauer chamber. Also, the pH and titratable acidity expressed in lactic acid were measured. The data obtained were converted to a logarithmic scale to apply the sigmoidal equations of Gompertz, Logistic, Modified Logistic and Weibull. The results of the kinetic modeling gave us that the modified Logistics and Logistics models presented a better fit compared to the rest. Likewise, the Weibull model presented the lowest value of adjustment, on the other hand, in the analysis of the statistical criteria, all models except Weibull present similarity. Finally, each sigmoidal model allowed to evaluate the growth of Saccharomyces boulardii with each of its kinetic constants.

Tailoring Au Films at the Atomic Scale with an Electron Beam Tweezer.

A thin, clean pristine Au film created in a transmission electron microscope chamber was tailored by an electron beam. Various kinds of nanopatterns, including hexagonal holes and dumbbell-like patterns, were fabricated by different doses of the electron beam. A high-quality series of in situ images were recorded to explore the irradiation mechanism. The electron-matter collision enabled the electron beam to act as a tweezer to arrange atoms into a specified pattern.

Multi-Scale Digital Image Correlation Analysis of In Situ Deformation of Open-Cell Porous Ultra-High Molecular Weight Polyethylene Foam.

Porous ultra-high molecular weight polyethylene (UHMWPE) is a high-performance bioinert polymer used in cranio-facial reconstructive surgery in procedures where relatively low mechanical stresses arise. As an alternative to much stiffer and more costly polyether-ether-ketone (PEEK) polymer, UHMWPE is finding further wide applications in hierarchically structured hybrids for advanced implants mimicking cartilage, cortical and trabecular bone tissues within a single component. The mechanical behaviour of open-cell UHMWPE sponges obtained through sacrificial desalination of hot compression-moulded UHMWPE-NaCl powder mixtures shows a complex dependence on the fabrication parameters and microstructural features. In particular, similarly to other porous media, it displays significant inhomogeneity of strain that readily localises within deformation bands that govern the overall response. In this article, we report advances in the development of accurate experimental techniques for operando studies of the structure–performance relationship applied to the porous UHMWPE medium with pore sizes of about 250 µm that are most well-suited for live cell proliferation and fast vascularization of implants. Samples of UHMWPE sponges were subjected to in situ compression using a micromechanical testing device within Scanning Electron Microscope (SEM) chamber, allowing the acquisition of high-resolution image sequences for Digital Image Correlation (DIC) analysis. Special masking and image processing algorithms were developed and applied to reveal the evolution of pore size and aspect ratio. Key structural evolution and deformation localisation phenomena were identified at both macro- and micro-structural levels in the elastic and plastic regimes. The motion of pore walls was quantitatively described, and the presence and influence of strain localisation zones were revealed and analysed using DIC technique.

A novel experimental set-up for in-situ microstructural characterization during continuous strain path change

Strain path change is a typical phenomenon during continuous stamping operations of sheet metal for a variety of applications including automotive body parts. During stamping, a punch continuously deforms a metal sheet to produce a desired geometry while following various strain path transitions depending on overall design of the stamping process. The strain path change can potentially alter the expected forming limit of the material. Previous researchers investigated the effect of changing strain path by loading sample in two distinct steps. Typically, between the steps the sample is unloaded before being re-loaded in the new strain path. This practice reflects the key challenge in elucidating this strain path dependent deformation, which is the ability to control the strain path change in a single deformation stage in an experimental set-up. In this work, a novel testing rig and specimen geometry that is capable of changing the strain path of a sample continuously without unloading the specimen were conceptualised, modelled and subsequently manufactured. Using this apparatus, the specimen was deformed in the uniaxial strain path in the first step before being deformed biaxially without unloading in between the steps. Thus, the apparatus ensures that the sample undergoes a continuous strain path change without unloading between the steps. The size of this mechanical test rig permits it to be placed inside a scanning electron microscope (SEM) chamber in order to study strain path transition in-situ to highlight strain localization and related microstructural changes in real time. Utilizing this test set-up, strain path change and corresponding strain values along each strain path were evaluated. The changes in material microstructure were concurrently investigated using in-situ SEM and electron back scattered diffraction (EBSD) analysis.

Li4Br(OH)3 microstructure monitoring over its synthesis to tackle the lithium-based salts exploitation challenges as advanced phase change materials for storage technologies

How to overcome fastly and reliably the challenges to foster the lithium-based salts exploitation for latent heat storage technologies? In situ and real time microscopy is used to understand the discrepancies between the theoretical and experimental macroscopic properties of materials via the microscopic mechanisms. The feasibility of this method on the inorganic lithium salts is demonstrated despite their air/moisture-sensitivity and the common belief pretending that LiOH cannot be used for the synthesis of new materials inside the microscope chamber due to its decomposition in dry environment or under vacuum. The deviation source of ~30% from the theoretical energy density of 434 kWh/m3 has been investigated through the case study of Li4Br(OH)3, an uncommon promising phase change material. The hydration/dehydration of the starting materials appears as one of the main parameters, with applied temperature protocols, eliciting the deviation towards different materials from the targeted one of interest, in terms of morphology and properties. This criterion, if not taken into account, could be disastrous for the storage capacity of a unit during its use. This study highlights solutions to avoid these deficiencies. The results consistency at microscale with those obtained at macroscale is also proved despite the different operating conditions

Environmental Scanning Electron Microscopy of Dermal Fibroblasts on Various Types of Polymer Scaffolds

With the progress in tissue engineering, the need for deep understanding of the interaction between individual cells and cellular systems with biocompatible polymer scaffolds has become obvious. This requires data on the adhesion and proliferation of cells on the scaffolds. Environmental scanning electron microscopy makes it possible to increase the pressure and humidity in a microscope chamber, thereby bringing the experimental conditions close to environmental. Scanning electron microscopy study of dermal fibroblasts is illustrated by the example of several scaffolds of different morphologies, including sponges, films, and nonwoven materials based on poly(L-)lactide, polycaprolactone, and a poly(L-)lactide-co-polycaprolactone. The data obtained show that the proposed method is promising for studying cell structures on polymer scaffolds.

Abstract 2714: The metastatic potential of circulating cancer spheroids: Diagnostic assay development using embryonic circulation models

Abstract Introduction: Progression into metastatic disease is a critical step in most malignancies. Cancer cells may enter circulation very early (circulating tumor cells or CTCs), but distant metastases are disseminated only by cancer stem cells (CSCs) that survive circulation. CSCs are known to form cell clusters in vitro (spheroids/tumorspheres). We reported that tumorspheres also circulate in blood in vivo. We found that they carry cancer stem cells and strongly correlate with metastatic disease. We collected tumorspheres from blood by filtration. This project aims to test the metastatic potential of tumorspheres by developing an assay using in vivo circulation models. The goal is to establish a novel prognostic test for metastatic predisposition in cancer patients. Methods: To develop an in vivo assay for metastasis we use the chorioallantoic circulation in the chicken embryo, with several advantages: easy access to circulation, lack of immune response, rapid turnaround time (5 to 10 days) and inexpensive and simple technique. For clinical applications we introduced three technical innovations: (i) to make fertilized chicken embryos easily available we worked out a method to store viable embryos beyond 30 days; (ii) we developed a new coverglass chamber technique for micromanipulations; and (iii) for microscopic analysis new over-drained chorioallantois preparations can accommodate high resolution objectives. To test tumorspheres in embryonic circulation we used both highly metastatic (e.g. MDA-MB-436) and non-metastatic (e.g. MCF7) cell lines with GFP label, DiI membrane dye and CFDA-SA dye. For molecular studies DNA was extracted 5 days after cancer spheroid implantation and used qPCR to detect human sequences. Results: In traditional storage few embryos survive beyond 6 days. As fertilized eggs carry pluripotent stem cells, we optimized temperature, gas and humidity combinations to match known stem cell optimums. By suppressing metabolism, blocking embryo adhesion and controlling gas and humidity exchange we extended viable embryo storage beyond 30 days. We modified the traditional window method by forming visually clear microscopic coverglass chambers, and the new over-draining approach lowered the chorioallantois membranes and allowed high resolution imaging. The results showed invasive and hematogenic dissemination of the cancer spheroids. Molecular studies of human markers detected metastatic progression and generated quantitative data. Conclusions: We optimized the in vivo embryonic metastasis assay. We show that it has the potential for a sensitive and quantitative method to diagnose metastatic predisposition in cancer patients. The molecular approach (DNA extraction, PCR markers) is easily up-scalable, the instrumentation is available in most hospitals and offers cost effective and critical early diagnosis of cancer progression. Citation Format: Peter Geck, James Kubilus, Andrew Makarovskiy, Kathleen Marinelli, Christine Kyritsis, Viktoria Denes. The metastatic potential of circulating cancer spheroids: Diagnostic assay development using embryonic circulation models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2714.