Correlative Microscopy Research Articles

Bringing atom probe tomography to transmission electron microscopes.

For the purpose of enhancing the structural insights within the three-dimensional composition fields revealed by atom probe tomography, correlative microscopy approaches, combining (scanning) transmission electron microscopy with atom probe tomography, have emerged and demonstrated their relevance. To push the boundaries further and facilitate a more comprehensive analysis of nanoscale matter by coupling numerous two- or three-dimensional datasets, there is an increasing interest in combining transmission electron microscopy and atom probe tomography into a unified instrument. This study presents the tangible outcome of an instrumental endeavour aimed at integrating atom probe tomography into a commercial transmission electron microscope. The resulting instrument demonstrates the feasibility of combining in situ 3D reconstructions of composition fields with the detailed structural analysis afforded by transmission electron microscopy. This study shows a promising approach for converging these two important nanoscale microscopy techniques.

Correlative light and soft X-ray tomography of in situ mesoscale heterochromatin structure in intact cells.

Heterochromatin organization is critical to many genome-related programs including transcriptional silencing and DNA repair. While super-resolution imaging, electron microscopy, and multiomics methods have provided indirect insights into the heterochromatin organization, a direct measurement of mesoscale heterochromatin ultrastructure is still missing. We use a combination of correlative light microscopy and cryo-soft X-ray tomography (CLXT) to analyze heterochromatin organization in the intact hydrated state of human mammary fibroblast cells. Our analysis reveals that the heterochromatin ultra-structure has a typical mean domain size of approximately 80nm and a mean separation of approximately 120nm between domains. Functional perturbations yield further insights into the molecular density and alterations in the mesoscale organization of the heterochromatin regions. Furthermore, our polymer simulations provide a mechanistic basis for the experimentally observed size and separation distributions of the mesoscale chromatin domains. Collectively, our results provide direct, label-free observation of heterochromatin organization in the intact hydrated state of cells.

A Cryo‐to‐Liquid Phase Correlative Light Electron Microscopy Workflow for the Visualization of Biological Processes in Graphene Liquid Cells

AbstractLiquid phase electron microscopy (LP‐EM) has emerged as a powerful technique for in situ observation of material formation in liquid. Especially the use of graphene as window material provides new opportunities to image biological processes because of graphene's molecular thickness and electron scavenger capabilities. However, in most cases the process of interest is initiated when the graphene liquid cells (GLCs) are sealed, meaning that the process cannot be imaged at early timepoints. Here, a novel cryogenic/liquid phase correlative light/electron microscopy workflow that addresses the delay time between graphene encapsulation and the start of the imaging, while combining the advantages of fluorescence and electron microscopy is reported. This workflow allows imaging to be initiated at a predetermined space and time by vitrifying and thawing at a selected time point. The workflow is demonstrated first by observing multiple day crystallization processes and subsequently highlight its potential by observing a biological process: the complexation of calciprotein particles. The ability to correlate the dynamic complexation observed in a GLC with cryogenic TEM and dynamic light scattering, confirms the validity of observations and underlines the exciting possibilities for LP‐EM in biology.

Elucidating the Nature of Secondary Phases in LiNi0.5Mn1.5O4 Cathode Materials using Correlative Raman-SEM Microscopy

Elucidating the Nature of Secondary Phases in LiNi0.5Mn1.5O4 Cathode Materials using Correlative Raman-SEM Microscopy

An Integrated Approach to Elucidate the Interplay between Iron Uptake Dynamics and Magnetosome Formation at the Single-Cell Level in Magnetospirillum gryphiswaldense.

Iron is a crucial element integral to various fundamental biological molecular mechanisms, including magnetosome biogenesis in magnetotactic bacteria (MTB). Magnetosomes are formed through the internalization and biomineralization of iron into magnetite crystals. However, the interconnected mechanisms by which MTB uptake and regulate intracellular iron for magnetosome biomineralization remain poorly understood, particularly at the single-cell level. To gain insights we employed a holistic multiscale approach, i.e., from elemental iron species to bacterial populations, to elucidate the interplay between iron uptake dynamics and magnetosome formation in Magnetospirillum gryphiswaldense MSR-1 under near-native conditions. We combined a correlative microscopy approach integrating light and X-ray tomography with analytical techniques, such as flow cytometry and inductively coupled plasma spectroscopy, to evaluate the effects of iron and oxygen availability on cellular growth, magnetosome biogenesis, and intracellular iron pool in MSR-1. Our results revealed that increased iron availability under microaerobic conditions significantly promoted the formation of longer magnetosome chains and increased intracellular iron uptake, with a saturation point at 300 μM iron citrate. Beyond this threshold, additional iron did not further extend the magnetosome chain length or increase total intracellular iron levels. Moreover, our work reveals (i) a direct correlation between the labile Fe2+ pool size and magnetosome content, with higher intracellular iron concentrations correlating with increased magnetosome production, and (ii) the existence of an intracellular iron pool, distinct from magnetite, persisting during all stages of biomineralization. This study offers insights into iron dynamics in magnetosome biomineralization at a single-cell level, potentially enhancing the industrial biomanufacturing of magnetosomes.

Unravelling the dominant influence of microsegregation on creep rupture behaviour of additively manufactured inconel 718

This investigation focuses on unravelling the dominant influence of microsegregation and microstructure altered due to heat treatment cycle variation on creep rupture behaviour of additively manufactured Inconel 718 (AM-IN718). Two microstructural variants differing in the fraction of recrystallized grains while δ-phase being absent, were produced. A typical heat treatment (HT) cycle includes the stress-relieving of the as-built specimens at 980 °C, followed by solution treatment at 1080 °C (STA1080, a partially recrystallized microstructural variant) or 1150 °C (STA1150, a fully recrystallized microstructural variant), and double ageing (soaking at 720 °C for 8h and subsequent furnace cooling, followed by 8h at 620 °C and air cooling).Detailed microstructural characterization of two microstructural variants through correlative microscopy revealed a prevalent existence of Nb-rich precipitate-free zones (PFZ) in STA1080 than in STA1150. Creep characterization of the two microstructural variants in the temperature range of 625–675 °C and at 500–750 MPa demonstrated superior creep resistance in STA1150. The correlation of kinetic analysis and comprehensive post-deformation microstructural characterization suggests grain boundary cavitation as the main damage/softening mechanism and the reason for the difference in creep rupture behaviour between the two microstructural variants. The long-term exposure heat treatment methodology demonstrates that PFZs are the major influencing factor responsible for microsegregation-dependent creep rupture behaviour. Interestingly, the presence of the δ phase within PFZs appeared to retard cavity coalescence and failure during creep, despite its usual detrimental role in creep resistance.

Stepwise virus assembly in the cell nucleus revealed by spatiotemporal click chemistry of DNA replication.

Biomolecular assemblies are fundamental to life and viral disease. The spatiotemporal coordination of viral replication and assembly is largely unknown. Here, we developed a dual-color click chemistry procedure for imaging adenovirus DNA (vDNA) replication in the cell nucleus. Late- but not early-replicated vDNA was packaged into virions. Early-replicated vDNA segregated from the viral replication compartment (VRC). Single object tracking, superresolution microscopy, fluorescence recovery after photobleaching, and correlative light-electron microscopy revealed a stepwise assembly program involving vDNA and capsid intermediates. Depending on replication and the scaffolding protein 52K, late-replicated vDNA with rapidly exchanging green fluorescent protein-tagged capsid linchpin protein V and incomplete virions emerged from the VRC periphery. These nanogel-like puncta exhibited restricted movements and were located with the capsid proteins hexon, VI, and virions in the nuclear periphery, suggestive of sites for virion formation. Our findings identify VRC dynamics and assembly intermediates, essential for stepwise productive adenovirus morphogenesis.

Correlative microscopy - illuminating the endomembrane system of plant seeds.

The endomembrane system of cereal seed endosperm is a highly plastic and dynamic system reflecting the high degree of specialization of this tissue. It is capable of coping with high levels of storage protein synthesis and undergoes rapid changes to accommodate these storage proteins in newly formed storage organelles such as endoplasmic reticulum-derived protein bodies or protein storage vacuoles. The study of endomembrane morphology in cereal endosperm is challenging due to the amount of starch that cereal seeds accumulate and the progressive desiccation of the tissue. Here, we present a comprehensive study of the endomembrane system of developing barley endosperm cells, complemented by correlative light and electron microscopy (CLEM) imaging. The use of genetically fused fluorescent protein tags in combination with the high resolution of electron microscopy brings ultrastructural research to a new level and can be used to generate novel insights in cell biology in general and in cereal seed research in particular.

Precision in situ cryogenic correlative light and electron microscopy of optogenetically positioned organelles.

Unambiguous targeting of cellular structures for in situ cryo-electron microscopy in the heterogeneous, dense and compacted environment of the cytoplasm remains challenging. Here, we have developed a cryogenic correlative light and electron microscopy (cryo-CLEM) workflow that utilizes thin cells grown on a mechanically defined substratum for rapid analysis of organelles and macromolecular complexes by cryo-electron tomography (cryo-ET). We coupled these advancements with optogenetics to redistribute perinuclear-localised organelles to the cell periphery, allowing visualisation of organelles that would otherwise be positioned in cellular regions too thick for cryo-ET. This reliable and robust workflow allows for fast in situ analyses without the requirement for cryo-focused ion beam milling. Using this protocol, cells can be frozen, imaged by cryo-fluorescence microscopy and be ready for batch cryo-ET within a day.

First person – Vikas Tillu

ABSTRACT First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping researchers promote themselves alongside their papers. Vikas Tillu is co-first author on ‘ Precision in situ cryogenic correlative light and electron microscopy of optogenetically positioned organelles’, published in JCS. Vikas is a Postdoctoral Researcher in the lab of Nicholas Ariotti at Institute for Molecular Bioscience, The University of Queensland, Australia, investigating in situ receptor trafficking analysis using cryo-electron tomography (cryo-ET).

Correlative analysis of advanced microscopy techniques for metallography and corrosion microstructures of bronze phoenician coins

Correlative microscopy stands out for its ability to concurrently acquire and analyze various data types in a multimodal and multiscale environment, enabling precise localization of specific areas within samples and enhancing the accuracy and relevance of analyses. This approach showed promise in revealing the metallurgical history of ancient coins. This article focused on composition, microstructure, and manufacturing process of Phoenician-Punic copper-based alloy coins (5th–4th century BCE). The four coins studied by correlative light and electron microscopy, μ-Raman spectroscopy, and X-ray Microscopy exhibited notable differences in elemental composition and microstructures. These variations are attributed to their origin from casting, followed by striking, and subsequent recrystallization due to a more intricate corrosion process.

Exploring Transient States of PAmKate to Enable Improved Cryogenic Single-Molecule Imaging.

Super-resolved cryogenic correlative light and electron microscopy is a powerful approach which combines the single-molecule specificity and sensitivity of fluorescence imaging with the nanoscale resolution of cryogenic electron tomography. Key to this method is active control over the emissive state of fluorescent labels to ensure sufficient sparsity to localize individual emitters. Recent work has identified fluorescent proteins (FPs) that photoactivate or photoswitch efficiently at cryogenic temperatures, but long on-times due to reduced quantum yield of photobleaching remain a challenge for imaging structures with a high density of localizations. In this work, we explore the photophysical properties of the red photoactivatable FP PAmKate and identify a 2-color process leading to enhanced turn-off of active emitters, improving localization rate. Specifically, after excitation of ground state molecules, we find that a transient state forms with a lifetime of ∼2 ms under cryogenic conditions, which can be bleached by exposure to a second wavelength. We measure the response of the transient state to different wavelengths, demonstrate how this mechanism can be used to improve imaging, and provide a blueprint for the study of other FPs at cryogenic temperatures.

Lift-Out Specimen Preparation and Multiscale Correlative Investigation of Li-Ion Battery Electrodes Using Focused Ion Beam-Secondary Ion Mass Spectrometry Platforms.

Advanced characterization is paramount to understanding battery cycling and degradation in greater detail. Herein, we present a novel methodology of battery electrode analysis, employing focused ion beam (FIB) secondary-ion mass spectrometry platforms coupled with a specific lift-out specimen preparation, allowing us to optimize analysis and prevent air contamination. Correlative microscopy, combining electron microscopy and chemical imaging of a liquid electrolyte Li-ion battery electrode, is performed over the entire electrode thickness down to subparticle domains. We observed a distinctive remnant lithiation among interparticles of the anode at the discharge state. Furthermore, chemical mapping reveals the nanometric architecture of advanced composite active materials with a lateral resolution of 16 nm and the presence of a solid electrolyte interface on the particle boundaries. We highlight the methodological advantages of studying interfaces and the ability to conduct high-performance chemical and morphological correlative analyses of battery materials and comment on their potential use in other fields.

Detection of Tn-antigen in breast and prostate cancer models by VVL-labeled red dye-doped nanoparticles.

Aim: Fluorescence detection of breast and prostate cancer cells expressing Tn-antigen, a tumor marker, with Vicia villosa lectin (VVL)-labeled nanoparticles.Materials & methods: Breast and prostate cancer cells engineered to express high levels of Tn-antigen and non-engineered controls were incubated with VVL-labeled or unlabeled red dye-doped silica-coated polystyrene nanoparticles. The binding to cells was studied with flow cytometry, confocal microscopy, and electron microscopy.Results: Flow cytometry showed that the binding of VVL-labeled nanoparticles was significantly higher to Tn-antigen-expressing cancer cells than controls. Confocal microscopy demonstrated that particles bound to the cell surface. According to the correlative light and electron microscopy the particles bound mostly as aggregates.Conclusion: VVL-labeled nanoparticles could provide a new tool for the detection of Tn-antigen-expressing breast and prostate cancer cells.

ARF1 compartments direct cargo flow via maturation into recycling endosomes.

Cellular membrane homoeostasis is maintained via a tightly regulated membrane and cargo flow between organelles of the endocytic and secretory pathways. Adaptor protein complexes (APs), which are recruited to membranes by the small GTPase ARF1, facilitate cargo selection and incorporation into trafficking intermediates. According to the classical model, small vesicles would facilitate bi-directional long-range transport between the Golgi, endosomes and plasma membrane. Here we revisit the intracellular organization of the vesicular transport machinery using a combination of CRISPR-Cas9 gene editing, live-cell high temporal (fast confocal) or spatial (stimulated emission depletion) microscopy as well as correlative light and electron microscopy. We characterize tubulo-vesicular ARF1 compartments that harbour clathrin and different APs. Our findings reveal two functionally different classes of ARF1 compartments, each decorated by a different combination of APs. Perinuclear ARF1 compartments facilitate Golgi export of secretory cargo, while peripheral ARF1 compartments are involved in endocytic recycling downstream of early endosomes. Contrary to the classical model of long-range vesicle shuttling, we observe that ARF1 compartments shed ARF1 and mature into recycling endosomes. This maturation process is impaired in the absence of AP-1 and results in trafficking defects. Collectively, these data highlight a crucial role for ARF1 compartments in post-Golgi sorting.

Experimental and analytical study on the compressive behavior of PMI foam with different densities and cell sizes under intermediate strain rates

Polymethacrylimide (PMI) foam materials have great potential for applications in the field of protective energy absorption owing to their superior compressive properties. Existing studies on the mechanical behavior of PMI foams are limited, particularly for loading at intermediate strain rates. This paper presents a comprehensive experimental study of PMI foam with four different densities under quasi-static and intermediate strain rate compression (0.001 s−1 to 100 s−1). Each density included three different cell sizes to determine their effects on the compressive performance. Loads parallel and perpendicular to the foam rise direction were considered to investigate the anisotropic behavior. Intermediate strain rate tests were conducted using a high-speed hydraulic servo testing machine, which achieved a stable strain rate while ensuring consistent specimen sizes in both quasi-static and dynamic tests. In all the experiments, the compression process was captured using a high-speed camera and the macroscopic deformation mode and microscopic deformation mechanism were analyzed by combining Digital Image Correlation (DIC) and Scanning Electron Microscopy (SEM). The PMI foam with finer cell sizes exhibited an enhanced compression performance. As the strain rate increased, the strain rate effect became more evident in the high-density specimens and an increase in cell size diminished this effect. A modified analytical model incorporating the cell-size correction term was developed based on Gibson and Ashby's model. The modified model exhibited an average error of 4.9 % in the predicted plateau stress of PMI foam with different cell sizes at the same density.

Unveiling the oxidation stability of 2D gallium-intercalated monolayer epitaxial graphene through correlative microscopy

The oxidation- and air-stability of 2D gallium-intercalated monolayer epitaxial graphene was determined using correlative microscopy. Site-specific studies including AFM, scanning electron microscope, cross section STEM-HAADF, and EELS revealed that the oxygen signal detected by XPS and AES analyses originated from oxidized surface carbon contaminants without the presence of oxygen at the 2D gallium layers. In addition, the air-stability of the 2D gallium was correlated with the presence of intact epitaxial graphene. The absence of graphene leads to oxidation of the 2D gallium in air, consequently losing the crystallinity of the epitaxial gallium layer. This study invokes the importance of correlative microscopy to better understand defects in 2D metals that have been recently recognized through the confinement heteroepitaxy. In addition, this study highlights the advantage of using high spatial resolution STEM techniques in comparison with XPS that has relatively lower resolution.

Localization of albumin with correlative super resolution light- and electron microscopy in the kidney

The functioning of vertebrate life relies on renal filtration of surplus fluid and elimination of low-molecular-weight waste products, while keeping serum proteins in the blood. In disease, however, there is leak of serum proteins and tracing them to identify the leaking position within tissue with a nanometer resolution poses a significant challenge. Correlative microscopy integrates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Using chemical tagging of albumin with synthetic fluorophores we achieve protein-specific labeling that preserve their post-embedding fluorescence after high-pressure freezing and freeze-substitution of murine kidney tissue. Using advanced registration techniques for super-resolution correlative light and electron microscopy, we can localize the labeled albumin with a high precision in the x-y plane of electron micrographs and cartograph its distribution. Thereby we can quantify the albumin concentration and measure a linear reduction gradient across the kidney filtration barrier. Our study shows the feasibility of combining different microscopy contrasts for tracing fluorescently labeled protein markers with super resolution in various tissue samples and opens new perspectives for correlative imaging in volume electron microscopy.

Correlative Raman spectroscopy and electron microscopy identifies glycogen rich deposits correlated with local structural defects in long bones of type IV osteogenesis imperfecta patients

Osteogenesis imperfecta (OI) is a genetic bone disease occurring in approximately 1 in 10,000 births, usually as a result of genetic mutation. OI patients suffer from increased fracture risk and - depending on the severity of the disease - deformation of the limbs, which can even lead to perinatal death.Despite extensive studies, the way in which the genetic mutation is translated into structural and compositional anomalies of the tissue is still an open question. Different observations have been reported, ranging from no structural (or chemical) differences to completely chaotic bone structure and composition.Here, we investigated bone samples from two adolescent OI-IV patients, focusing on the bone structure and chemistry in naturally occurring fractures. The exposed fracture plane allows the investigation of the structure and composition of the weakest bone plane. We do so by combining scanning electron microscopy (SEM) imaging with chemical information from Raman microscopy.The exposed fracture planes show different regions within the same tissue, displaying normal osteonal structures next to disorganized osteons and totally disordered structures, while the collagen mineralization in all cases is similar to that of a healthy bone.In addition, we also detected significant amounts of depositions of glycogen-rich, organic, globules of 250-1000nm in size. These depositions point to a role of cellular disfunction in the disorganization of the collagen in qualitative OI.Overall, our results unite multiple, sometimes contradicting views from the literature on qualitative OI.

Flexural properties of fiber-reinforced concrete using hybrid recycled steel fibers and manufactured steel fibers

This study investigates the flexural properties of fiber-reinforced concrete (FRC) using a hybrid mix of recycled steel fibers (RSF) from waste tires and manufactured steel fibers (MSF). The aim of this study is to demonstrate hybrid steel fiber reinforcement as a more environmentally sustainable alternative to traditional single-fiber systems, by assessing whether hybrid reinforcement can achieve or surpass the performance of conventional single-manufactured steel fiber reinforcement. Using a novel four-stage methodology, this paper presents a comprehensive and detailed evaluation of mechanical and ductility performance through three-point bending tests, digital image correlation (DIC), CT-scans, and scanning electron microscopy (SEM). The results from flexural stress-deflection curves were used to evaluate the flexural strength and toughness. This was followed by an in-depth eight-stage DIC analysis and Surface Damage Index (SDI) curves throughout the test. CT-scan images were used to quantify the proportion of fibers classified as pulled-out, ruptured, or bridging the crack, while SEM analyzed the efficiency of fiber reinforcement through microstructural evaluations, further validating the findings of this study. Results show that hybrid reinforcement significantly enhances both toughness and ductility while reducing surface damage. MSFs in the hybrid system improve post-peak ductility and energy absorption by bridging large cracks, while RSFs prevent micro-cracks, increasing pre-peak rigidity. CT-scan analysis reveals that the inclusion of RSFs reduces pulled-out MSFs by 26%, further boosting flexural performance. SEM confirms the superior bond of MSFs with the concrete matrix in hybrid samples. Additionally, higher fiber content in FRC enhances toughness and ductility while reducing surface damage. This research advances our understanding of hybrid fiber-reinforced concrete and promotes more sustainable construction practices through the use of recycled steel fibers. By leveraging the combination of new methodologies, the study effectively targets gaps in the current research landscape, offering a comprehensive approach to improving performance and sustainability in construction materials.