Correlative Microscopy Approach Research Articles

A correlative approach to evaluating the links between local microstructural parameters and creep initiated cavities

The study and modelling of material degradation processes, such as the initiation and growth of creep cavities in high-temperature applications, require a correlative and comprehensive knowledge of the microstructure. However, individual microscopy is limited to a small region and specific microstructural information of the specimen. This work demonstrates a novel correlative microscopy approach for characterising creep cavitation and establishing correlations with local microstructural parameters in a statistical manner. This approach combines datasets from stitched higher-resolution backscattered electron (BSE) images, XeF2 Focused Ion Beam (FIB) images, and backscattered electron diffraction (EBSD) maps with advanced image correlation techniques. Deep-learning image segmentation techniques and statistical analysis are applied to find relations between creep cavitation and local microstructural environment. This approach is demonstrated in a cyclic creep-tested 316H stainless steel specimen with extensive creep cavities. The results show that in this material, strain localization, grain boundary misorientation, and substantial precipitation dominate the nucleation of cavities, whereas other microstructural properties such as grain size and Schmid factor play smaller roles. This study presents the use of the correlative microscopy approach to provide new insights into creep cavitation behaviour and its implications for establishing creep cavitation damage models.

A multi-scale microscopic study of phase transformations and concomitant α/β interface evolution in additively manufactured Ti-6Al-4V alloy

Ti-6Al-4V alloy produced by laser-based powder bed fusion (LPBF) technique consists of an α′-martensite phase which is usually brittle and hence required, microstructure optimization by heat treatment to improve ductility. In this study, detailed microstructure analysis in the as-built condition and its evolution after sub-transus annealing was investigated. On annealing, the α′-martensite decomposes into α- and β-phases with the evolution of the α/β interface. Considering that the composition and microstructure play a crucial role in deciding the mechanical properties and service performance, a multi-scale correlative microscopy approach involving Transmission Electron Microscopy (TEM), Transmission Kikuchi Diffraction (TKD), and Atom Probe Tomography (APT) was employed. In the as-built condition, predominant segregation of β-stabilizers at the defect site especially along grain boundaries was observed; and these act as potential nucleation sites for β-phase during subsequent annealing. Low temperature annealing (750ᵒC) transforms the V-rich regions into the β-phase in the form of nanoscale precipitates. However, as the annealing temperature increases above 800ᵒC, the β-phase adopts a rod-like morphology. Comparing α-stabilizers, it is realized that β-stabilizers have a twofold greater contribution to elemental partition. Further, the composition and width of the α/β interface significantly impacts the mechanical properties of the alloy. Annealing at 750 °C and 850 °C resulted in significant improvement in ductility by 57.5% and 62.7% respectively without much of a trade-off in strength (>1 GPa). The strength and ductility are seen to decline when annealing temperature is raised above 850 °C due to a rise in compositional fluctuation and broadening of the α/β interface.

Grain size effect on the phase growth in CoNi/Sn sandwich diffusion couples

Grain size effect during interdiffusion is investigated by designing a sandwich couple of CG-CoNi/Sn/UFG-CoNi; (CG coarse grained (>200 µm), UFG: ultrafine grained (∼240 nm)). Phase growth is examined at temperatures 175 & 200 ℃ for various times. CG CoNi and UFG CoNi have been fabricated by Vacuum arc melting and spark plasma sintering of mechanically alloyed CoNi powders, respectively. At 175 ℃, (CoNi)Sn3 intermetallic phase is detected at CG CoNi/Sn interface and (CoNi)Sn2 phase at UFG CoNi/Sn interface. At 200℃, formation of (CoNi)Sn2 and (CoNi)Sn3 phases is observed at CG interface and only (CoNi)Sn2 at the UFG interface. Phase compositions have been confirmed by electron probe microanalysis while the corresponding crystal structure with micro-x-ray diffraction. Phase selection in the interdiffusion zone (IDZ) of CG CoNi/Sn is governed primarily by kinetic factors, while thermodynamic driving forces, assessed using CALPHAD approach, also contribute to phase formation in the IDZ of UFG CoNi/Sn. Time-dependent measurements at 175 & 200℃ for UFG CoNi/Sn reveal an accelerated growth of phases when compared with the CG CoNi/Sn. The enhanced kinetics and preferential formation of the (CoNi)Sn2 phase at the UFG CoNi/Sn interface can be ascribed to the higher proportion of grain boundaries (GBs), leads to an increase in overall diffusion flux. The sandwich diffusion couple approach ensured that all the comparisons between CG and UFG interfaces are conducted under similar conditions yielding dependable correlations. The formation of intermetallic phases and role of GBs are studied at a finer detail using correlative microscopy approach.

Exploring the infiltrative and degradative ability of Fusarium oxysporum on polyethylene terephthalate (PET) using correlative microscopy and deep learning

Managing the worldwide steady increase in the production of plastic while mitigating the Earth’s global pollution is one of the greatest challenges nowadays. Fungi are often involved in biodegradation processes thanks to their ability to penetrate into substrates and release powerful catabolic exoenzymes. However, studying the interaction between fungi and plastic substrates is challenging due to the deep hyphal penetration, which hinders visualisation and evaluation of fungal activity. In this study, a multiscale and multimodal correlative microscopy workflow was employed to investigate the infiltrative and degradative ability of Fusarium oxysporum fungal strain on polyethylene terephthalate (PET) fragments. The use of non-destructive high-resolution 3D X-ray microscopy (XRM) coupled with a state-of-art Deep Learning (DL) reconstruction algorithm allowed optimal visualisation of the distribution of the fungus on the PET fragment. The fungus preferentially developed on the edges and corners of the fragment, where it was able to penetrate into the material through fractures. Additional analyses with scanning electron microscopy (SEM), Raman and energy dispersive X-ray spectroscopy (EDX) allowed the identification of the different phases detected by XRM. The correlative microscopy approach unlocked a more comprehensive understanding of the fungus-plastic interaction, including elemental information and polymeric composition.

Cholla cactus wood (Cylindropuntia imbricata): Hierarchical structure and micromechanical properties

The Cholla cactus is a species of cacti that survives in arid environments and produces a unique mesh-like porous wood. In this article, we present a comprehensive investigation on the hierarchical structure and micromechanical properties of the Cholla cactus wood. Multiple approaches consisting of X-ray tomography, scanning electron microscopy, scanning probe microscopy, nanoindentation, and finite element simulations were used to gain insight into the structure, property, and design principles of the Cholla cactus wood. The microstructure of the Cholla cactus wood consists of different components, including vessels, rays, and fibers. In the present study, we quantitatively describe the structure of each of these wood components and their likely functions, both from the perspective of biological and mechanical behavior. Nanoindentation experiments revealed for the first time that the cell walls of the fibers exhibit stiffness and hardness higher than those of rays. Furthermore, the idea of making porous, thin-walled cylinders was abstracted from the design of vessel elements, and the structures inspired by this principle were studied in tensile and torsional loading conditions using finite element simulations. Finite element simulations revealed that the utilization of a larger volume of material to carry the load leads to an increase in toughness of these structures, and thus suggested that the pores should be architected to maximize the distribution of load. Statement of significanceThe Cholla cactus wood possess a unique hierarchical structure that enables it to thrive in arid environments. Our correlative microscopy approach reveals incredible strategies that individual wood components exhibit to enable the survival of Cholla cactus in extreme environments. The present work quantifies the microstructure and mechanical properties of this very interesting natural system. We further investigate a design principle inspired by the vessel elements, one of the wood components of Cholla cactus, using finite element simulations. The study presented here advances our understanding of the structural significance of Cholla cactus and potentially other desert plants and will further help design architected structural materials.

Metal Core-Shell Nanoparticle Supercrystals: From Photoactivation of Hydrogen Evolution to Photocorrosion.

Gas nanobubbles are directly linked to many important chemical reactions. While they can be detrimental to operational devices, they also reflect the local activity at the nanoscale. Here, supercrystals made of highly monodisperse Ag@Pt core-shell nanoparticles are first grown onto a solid support and fully characterized by electron microscopies and X-ray scattering. Supercrystals are then used as a plasmonic photocatalytic platform for triggering the hydrogen evolution reaction. The catalytic activity is measured operando at the single supercrystal level by high-resolution optical microscopy, which allows gas nanobubble nucleation to be probed at the early stage with high temporal resolution and the amount of gas molecules trapped inside them to be quantified. Finally, a correlative microscopy approach and high-resolution electron energy loss spectroscopy help to decipher the mechanisms at the origin of the local degradation of the supercrystals during catalysis, namely nanoscale erosion and corrosion.

A thrombus is formed by a gradient of platelet activation and procoagulant endothelium

BackgroundThe contribution of platelets in thrombosis within microcirculation has been extensively documented in the literature. We previously showed, in vivo, that platelet activation revealed by intracellular calcium mobilization was a crucial step in the growth of thrombi following laser-induced injury, a model of thromboinflammation. ObjectiveOur goal was to investigate the extent of platelet activation and the spatial distribution of platelets throughout a growing thrombus. MethodsWe employed a multimodal, correlative microscopy approach and computational biology to study the state of platelets on a growing thrombus obtained after a laser injury. ResultsWe observed a reversible intracellular platelet calcium mobilization that correlates with the time a platelet resides during thrombus growth. Our bioinformatics analysis displayed the following 3 distinct platelet subpopulations resident within a thrombus: (1) resting, (2) partially activated, and (3) “fully” activated platelets. The spatial distribution of the platelet subpopulations in the thrombus creates a double gradient in both the transversal and longitudinal axis, with the maximal percentage of fully activated platelets close to the site of injury. However, these activated platelets did not express negative phospholipids. The injured endothelium was identified to play a vital role in activating the blood coagulation cascade in this model of thrombosis. ConclusionFollowing a laser-induced injury, thrombi are formed by a gradient of activated platelets from the injury site to the periphery of the thrombus. These different activation states of platelets throughout the thrombi regulate the biomechanics of the thrombus. The injured endothelium, rather than platelets, was identified to play a key role in the activation of the blood coagulation cascade in this model of thromboinflammation.

Multimodal and multiscale investigation for the optimization of AlSi10Mg components made by powder bed fusion-laser beam

In recent years, there has been a growing interest in the use of additive manufacturing (AM) to fabricate metallic components with tailored microstructures and improved mechanical properties. One of the most promising techniques for the aerospace industry is powder bed fusion-laser beam (PBF-LB). This technique enables the creation of complex shapes and structures with high accuracy and repeatability, which is especially important for the aerospace industry where components require high precision and reliability. However, the impact of the PBF-LB process on microstructural features, such as the grain size distribution and porosity, remains an important area of research since it influences mechanical properties and performance of materials. In this study, a multimodal and multiscale correlative microscopy approach is used to investigate the microstructure of AlSi10Mg components made by PBF-LB. The study found that the correlative microscopy approach involving X-ray images with visual, chemical, and diffraction information coming from optical microscopy (OM), scanning electron microscopy (SEM) and electron backscatter diffraction (EBSD) is highly effective in reaching a more comprehensive understanding of the relationship between the fabrication process and the effective microstructure of PBF-LB fabricated components enabling the optimization of their performance for a wide range of applications.

Morphofunctional changes at the active zone during synaptic vesicle exocytosis.

Synaptic vesicle (SV) fusion with the plasma membrane (PM) proceeds through intermediate steps that remain poorly resolved. The effect of persistent high or low exocytosis activity on intermediate steps remains unknown. Using spray-mixing plunge-freezing cryo-electron tomography we observe events following synaptic stimulation at nanometer resolution in near-native samples. Our data suggest that during the stage that immediately follows stimulation, termed early fusion, PM and SV membrane curvature changes to establish a point contact. The next stage-late fusion-shows fusion pore opening and SV collapse. During early fusion, proximal tethered SVs form additional tethers with the PM and increase the inter-SV connector number. In the late-fusion stage, PM-proximal SVs lose their interconnections, allowing them to move toward the PM. Two SNAP-25 mutations, one arresting and one disinhibiting spontaneous release, cause connector loss. The disinhibiting mutation causes loss of membrane-proximal multiple-tethered SVs. Overall, tether formation and connector dissolution are triggered by stimulation and respond to spontaneous fusion rate manipulation. These morphological observations likely correspond to SV transition from one functional pool to another.

Synthesis and Characterization of Gd-Functionalized B4C Nanoparticles for BNCT Applications

Inorganic nanoparticles of boron-rich compounds represent an attractive alternative to boron-containing molecules, such as boronophenylalanine or boranes, for BNCT applications. This work describes the synthesis and biological activity of multifunctional boron carbide nanoparticles stabilized with polyacrylic acid (PAA) and a gadolinium (Gd)-rich solid phase. A fluorophore (DiI) was included in the PAA functionalization, allowing the confocal microscopy imaging of the nanoparticles. Analysis of the interaction and activity of these fluorescent Gd-containing B4C nanoparticles (FGdBNPs) with cultured cells was appraised using an innovative correlative microscopy approach combining intracellular neutron autoradiography, confocal, and SEM imaging. This new approach allows visualizing the cells, the FGdBNP, and the events deriving from the nuclear process in the same image. Quantification of 10B by neutron autoradiography in cells treated with FGdBNPs confirmed a significant accumulation of NPs with low levels of cellular toxicity. These results suggest that these NPs might represent a valuable tool for achieving a high boron concentration in tumoral cells.

Correlative microscopy approach for micro- and nanoplastics detection in plant matrix

The world-wide plastics pollution into nature enters the human food chain as degradation products, influencing environmental habitat and health. Micro- and nanoplastics as foreign bodies contaminating beverages and foods on the cellular level are likely to have a long-term impact on nutrition growth, public and personal health. Nanoparticles and their spatial location are generally difficult to detect in biological specimens, especially plastic particles due to their analogous hydrocarbon-based matrix. Reliable analysis methods for the discovery of micro- and nanoplastic particles in entities are not fully established yet. We are therefore developing a correlative workflow for detecting plastics residuals in cellular tissue. We determined that light microscopy (LM) is usable for a coarse estimation of the contamination grade of plant samples. Due to its optical diffraction limit, LM rather detects nanoparticle patches than single particles, shown with fluorescent particles. Scanning electron microscopy (SEM) instead provides higher resolution, especially for single particle detection. EM sample staining is still required to depict polymerous nanoparticle specimens. Using LM and SEM data in a CLEM approach with TESCAN CORAL, discovery of contaminated plant areas is possible with single nanoparticle resolution. It reveals that nanoparticles seem to be tightly attached to the plant material. It indicates further that basic food cleaning procedures might be insufficient for particle removal. The workflow circumvents correlative sample 3D-topography issues. But insights into the plant matrix is best possible with z-information. Therefore, we additionally use focused ion beam (FIB) to determine inclusions of micro- and nanoplastics in the tissue/cellular matrix, investigating the optimal procedural approach as model for biomaterial systems. Procedural correlative microscopy provides a promising analysis method for the semiquantitative analysis of micro- and nanoparticles plant contaminations.

Correlative light and electron microscopic observation of calcium phosphate particles in a mouse kidney formed under a high-phosphate diet

Calcium phosphate forms particles under excessive urinary excretion of phosphate in the kidney. While the formation of calcium phosphate particles (CaPs) has been implicated in the damage to renal tubular cells and renal dysfunction, clarifying the ultrastructural information and the elemental composition of the small CaPs in the wide areas of kidney tissue has been technically difficult. This study introduces correlative and sequential light as well as electron microscopic CaP observation in the kidney tissue by combining fluorescent staining for CaPs and energy-dispersive X-ray spectroscopy (EDS) in scanning electron microscopy (SEM) on resin sections prepared using high-pressure freezing and freeze substitution. CaPs formed in mouse kidneys under long-term feeding of a high-phosphate diet were clearly visualized on resin sections by fluorescence-conjugated alendronate derivatives and toluidine blue metachromasia. These CaPs were verified by correlative observation with EDS. Furthermore, small CaPs formed in the kidney under short-term feeding were detected using fluorescent probes. The elemental composition of the particles, including calcium and magnesium, was identified following EDS analyses. These results suggest that the correlative microscopy approach is helpful for observing in situ distribution and elemental composition of CaPs in the kidney and contributing to studies regarding CaP formation-associated pathophysiology.

Correlative Light and Electron Cryo-Microscopy Workflow Combining Micropatterning, Ice Shield, and an In-Chamber Fluorescence Light Microscope.

In situ cryo-electron tomography (cryo-ET) is the most current, state-of-the-art technique to study cell machinery in its hydrated near-native state. The method provides ultrastructural details at sub-nanometer resolution for many components within the cellular context. Making use of recent advances in sample preparation techniques and combining this method with correlative light and electron microscopy (CLEM) approaches have enabled targeted molecular visualization. Nevertheless, the implementation has also added to the complexity of the workflow and introduced new obstacles in the way of streamlining and achieving high throughput, sample yield, and sample quality. Here, we report a detailed protocol by combining multiple newly available technologies to establish an integrated, high-throughput, optimized, and streamlined cryo-CLEM workflow for improved sample yield. Key features • PRIMO micropatterning allows precise cell positioning and maximum number of cell targets amenable to thinning with cryo focused-ion-beam-scanning electron microscopy. • CERES ice shield ensures that the lamellae remain free of ice contamination during the batch milling process. • METEOR in-chamber fluorescence microscope facilitates the targeted cryo focused-ion-beam (cryo FIB) milling of these targets. • Combining the three technologies into one cryo-CLEM workflow maximizes sample yield, throughput, and efficiency. Graphical overview.

Targeting Ultrastructural Events at the Graft Interface of Arabidopsis thaliana by A Correlative Light Electron Microscopy Approach.

Combining two different plants together through grafting is one of the oldest horticultural techniques. In order to survive, both partners must communicate via the formation of de novo connections between the scion and the rootstock. Despite the importance of grafting, the ultrastructural processes occurring at the graft interface remain elusive due to the difficulty of locating the exact interface at the ultrastructural level. To date, only studies with interfamily grafts showing enough ultrastructural differences were able to reliably localize the grafting interface at the ultrastructural level under electron microscopy. Thanks to the implementation of correlative light electron microscopy (CLEM) approaches where the grafted partners were tagged with fluorescent proteins of different colors, the graft interface was successfully and reliably targeted. Here, we describe a protocol for CLEM for the model plant Arabidopsis thaliana , which unambiguously targets the graft interface at the ultrastructural level. Moreover, this protocol is compatible with immunolocalization and electron tomography acquisition to achieve a three-dimensional view of the ultrastructural events of interest in plant tissues. Graphical abstract.

Serial-section Electron Tomography and Quantitative Analysis of Microtubule Organization in 3D-reconstructed Mitotic Spindles.

For the analysis of cellular architecture during mitosis, nanometer resolution is needed to visualize the organization of microtubules in spindles. Here, we present a detailed protocol that can be used to produce 3D reconstructions of whole mitotic spindles in cells grown in culture. For this, we attach mammalian cells enriched in mitotic stages to sapphire discs. Our protocol further involves cryo-immobilization by high-pressure freezing, freeze-substitution, and resin embedding. We then use fluorescence light microscopy to stage select mitotic cells in the resin-embedded samples. This is followed by large-scale electron tomography to reconstruct the selected and staged mitotic spindles in 3D. The generated and stitched electron tomograms are then used to semi-automatically segment the microtubules for subsequent quantitative analysis of spindle organization. Thus, by providing a detailed correlative light and electron microscopy (CLEM) approach, we give cell biologists a toolset to streamline the 3D visualization and analysis of spindle microtubules (http://kiewisz.shinyapps.io/asga). In addition, we refer to a recently launched platform that allows for an interactive display of the 3D-reconstructed mitotic spindles (https://cfci.shinyapps.io/ASGA_3DViewer/). Key features • High-throughput screening of mitotic cells by correlative light and electron microscopy (CLEM). • Serial-section electron tomography of selected cells. • Visualization of mitotic spindles in 3D and quantitative analysis of microtubule organization.

Microsecond melting and revitrification of cryo samples with a correlative light-electron microscopy approach.

We have recently introduced a novel approach to time-resolved cryo-electron microscopy (cryo-EM) that affords microsecond time resolution. It involves melting a cryo sample with a laser beam to allow dynamics of the embedded particles to occur. Once the laser beam is switched off, the sample revitrifies within just a few microseconds, trapping the particles in their transient configurations, which can subsequently be imaged to obtain a snap shot of the dynamics at this point in time. While we have previously performed such experiments with a modified transmission electron microscope, we here demonstrate a simpler implementation that uses an optical microscope. We believe that this will make our technique more easily accessible and hope that it will encourage other groups to apply microsecond time-resolved cryo-EM to study the fast dynamics of a variety of proteins.

Sensing Capabilities of Single Nanowires Studied with Correlative In Situ Light and Electron Microscopy.

Modern devices based on modular designs require versatile and universal sensor components which provide an efficient, sensitive, and compact measurement unit. To improve the space capacity of devices, miniaturized building elements are needed, which implies a turning away from conventional microcantilevers toward nanoscale cantilevers. Nanowires can be seen as high-quality resonators and offer the opportunity to create sensing devices on small scales. To use such a one-dimensional nanostructure as a resonant cantilever, a precise characterization based on the fundamental properties is needed. We present a correlative electron and light microscopy approach to characterize the pressure and environment sensing capabilities of single nanowires by analyzing their resonance behavior in situ. The high vacuum in electron microscopes enables the characterization of the intrinsic vibrational properties and the maximum quality factor. To analyze the damping effect caused by the interaction of the gas molecules with the excited nanowire, the in situ resonance measurements have been performed under non-high-vacuum conditions. For this purpose, single nanowires are mounted in a specifically designed compact gas chamber underneath the light microscope, which enables direct observation of the resonance behavior and evaluation of the quality factor with dependence of the applied gas atmosphere (He, N2, Ar, Air) and pressure level. By using the resonance vibration, we demonstrate the pressure sensing capability of a single nanowire and examine the molar mass of the surrounding atmosphere. Together this shows that even single nanowires can be utilized as versatile nanoscale gas sensors.

Atomic-Scale Characterization of Planar Selective-Area-Grown InAs/InGaAs Nanowires.

Atomic-scale information about the structural and compositional properties of novel semiconductor nanowires is essential to tailoring their properties for specific applications, but characterization at this length scale remains a challenging task. Here, quasi-1D InAs/InGaAs semiconductor nanowire arrays were grown by selective area epitaxy (SAE) using molecular beam epitaxy (MBE), and their subsequent properties were analyzed by a combination of atom probe tomography (APT) and aberration-corrected transmission electron microscopy (TEM). Results revealed the chemical composition of the outermost thin InAs layer, a fine variation in the indium content at the InAs/InGaAs interface, and lightly incorporated element tracing. The results highlight the importance of correlative microscopy approaches in revealing complex nanoscale structures, with TEM being uniquely suited to interrogating the crystallography of InGaAs NWs, whereas APT is capable of three-dimensional (3D) elemental mapping, revealing the subtle compositional variation near the boundary region. This work demonstrates a detailed pathway for the nanoscale structural assessment of novel one-dimensional (1D) nanomaterials.

Fingerprints Indicating Superior Properties of Internal Interfaces in Cu(In,Ga)Se2 Thin-Film Solar Cells.

Growth of Cu(In,Ga)Se2 (CIGS) absorbers under Cu-poor conditions gives rise to incorporation of numerous defects into the bulk, whereas the same absorber grown under Cu-rich conditions leads to a stoichiometric bulk with minimum defects. This suggests that CIGS absorbers grown under Cu-rich conditions are more suitable for solar cell applications. However, the CIGS solar cell devices with record efficiencies have all been fabricated under Cu-poor conditions, despite the expectations. Therefore, in the present work, both Cu-poor and Cu-rich CIGS cells are investigated, and the superior properties of the internal interfaces of the Cu-poor CIGS cells, such as the p-n junction and grain boundaries, which always makes them the record-efficiency devices, are shown. More precisely, by employing a correlative microscopy approach, the typical fingerprints for superior properties of internal interfaces necessary for maintaining a lower recombination activity in the cell is discovered. These are a Cu-depleted and Cd-enriched CIGS absorber surface, near the p-n junction, as well asa negative Cu factor (∆β) and high Na content (>1.5 at%) at the grain boundaries. Thus, this work provides key factors governing the device performance (efficiency), which can be considered in the design of next-generation solar cells.

New Correlative Microscopy Approaches to Understand the Microstructural Origins of Creep Cavitation in Austenitic Steels

New Correlative Microscopy Approaches to Understand the Microstructural Origins of Creep Cavitation in Austenitic Steels