A practical workflow for fixation and autofluorescence reduction in correlative light and electron microscopy of postmortem human brain tissue.

Cryo-scanning electron microscopy (CryoSEM) permits the preparation and detailed imaging of bulky samples while keeping them in a hydrated state. For plant biology, cryofractures give information on cell ultrastructure and tissue organisation within a much larger context that is the whole organ or organism. To date, a method to locate fluorescence reporters on the cryofracture has not been reported. Our approach uses a stereofluorescence microscope with an 80mm working distance and a high-zoom ratio to image the fracture through a viewing port of the cryopreparation chamber while the sample is still frozen and under vacuum. We have applied this method to look at fluorescent reporters of auxin transport and signalling in plant shoot apices and seedlings, the expression of a poorly characterised gene in the young floral pedicel and nitrogen-fixing rhizobial bacteria, expressing GFP, in root nodules. This method is applicable to any cryopreserved bulky sample that has a fluorescent output and paves the way for correlative light-electron microscopy for cryoSEM-based imaging.

The smut fungus Thecaphora frezzii causes severe yield losses in peanuts (Arachis hypogaea) in Argentina. Previous work established its fully intracellular biotrophic progression through subterranean organs and its exclusive sporulation within the seed coat, yet the ontogeny of teliospore formation in planta remained unresolved. Here, we applied a pragmatic correlative multiscale microscopy approach based on serial paraffin sections examined by stereomicroscopy, light microscopy, confocal laser scanning microscopy, and scanning electron microscopy, enabling spatial correlation of fungal structures within their tissue context. Using this integrative framework, we characterized the organization and progression of sporogenic structures associated with teliosporogenesis. Teliosporogenesis proved to be tightly synchronized with host tissue context and seed developmental stage, and was consistently preceded by a marked reorganization of sporogenous hyphae into three-dimensional coiled hyphal aggregates embedded in a mucilaginous matrix. These precursors undergo hyphal fragmentation followed by central-peripheral differentiation, whereby a small number of central units enlarge and individualize into teliospore initials while peripheral elements collapse, yielding stable teliospore balls as the final sporogenic product. This developmental sequence defines a distinct ontogenetic pattern not captured by current schemes of sporogenesis, here designated the Teliospore-ball type. Our results clarify the developmental pathways of T. frezzii sporulation in planta and demonstrate how accessible multiscale microscopy can be used to integrate structural information across spatial scales in complex plant-fungus interactions.

To elucidate the anatomical characteristics and three-dimensional continuity of a previously unrecognized thin-adipose compartment between the colonic mesentery and retroperitoneum, using correlative microscopy and block-face imaging. In this study, which was conducted at the anatomical laboratory of the Institute of Science Tokyo (formerly Tokyo Medical and Dental University), seven adult cadavers were examined. Histological analysis was conducted on six specimens using paraffin sections stained with Elastica van Gieson and Masson's trichrome. One cadaver underwent three-dimensional morphological analysis using correlative microscopy and block-face imaging. Serial block-face images of the perirenal region were captured at 100-μm intervals, and three-dimensional reconstruction segmentation was performed. A distinct thin-adipose compartment was consistently observed between the colonic mesentery and perirenal fat, enclosed by dense connective tissue and containing small vessels. Similar compartments were also found between the perirenal fat and pararenal fat, and beneath the peritoneum along the abdominal wall. These compartments extended in three directions from the peritoneal reflection and demonstrated craniocaudal continuity; laterally, these compartments converged to form a triad-like junction. The thin-adipose compartment represents a structurally organized anatomical unit rather than amorphous filler. Its consistent continuity and integration with adjacent structures support a compartment-based framework of intra-abdominal anatomy, which has potential relevance for understanding surgical anatomy.

Correlative microscopy has gained increasing importance across a range of research disciplines. Combining different microscopy techniques broadens knowledge about a sample by providing more comprehensive insights. In particular, the correlation of atomic force microscopy (AFM) and transmission electron microscopy (TEM) offers a powerful complementary approach for investigating materials, as both surface and subsurface information can be obtained. The fundamental motivation of this study is to establish a direct correlation between measurements obtained from the same specimen region by both methods. Such correlation is not always straightforward, as each technique requires different sample preparation. Consequently, performing AFM measurements on TEM samples inevitably gives rise to several challenges, including, but not limited to, surface distortion and limited accessibility. In this study, we propose a range of AFM measurement strategies tailored to two typical TEM sample types: a 3 nm thin membrane on lacey carbon and a TEM lamella mounted on a lift-out grid. We compare the influence of different cantilever dimensions and AFM modes on image quality, and explore the fabrication of AFM tips positioned at the very front of a cantilever via focused electron beam induced deposition to improve accessibility of regions of interest. With the strategies developed here, we successfully demonstrate the feasibility of AFM measurements on TEM samples without the need for additional sample preparation, enabling direct correlation. The results highlight the practical viability of this combined approach, and expand the scope of correlative microscopy for advanced materials characterization.

Cellular organelles are not just static structures; they are highly dynamic and directly linked to cellular functions. Changes in their morphology can be early indicators of diseases. Recent advancements in light microscopy techniques have transformed organelle research from qualitative descriptions to precise, quantitative measurements, enabling nanoscale resolution, high-throughput image analysis, and live-cell compatibility. This enables accurate measurement of organelle morphology, dynamics, and spatial organization using modern imaging and analysis techniques. By quantifying organelles, we go beyond simply visualizing to measuring and statistically comparing cellular features across different samples. This article addresses a wide range of cellular organelles across all major experimental systems, specifically mentioning mitochondria, myofibers, actin filaments, endoplasmic reticulum, and Golgi apparatus, by integrating experimental design, optimized sample preparation, high-resolution imaging, and validated Fiji/ImageJ-based analysis workflows. For each organelle, step-by-step protocols specify reagents, equipment, acquisition parameters, and expected results. Although recent advances, such as expansion microscopy, correlative light-electron microscopy, and AI-powered segmentation, offer gains in throughput and resolution, this workflow demonstrates that Fiji-based analysis remains fully capable of delivering high-precision organelle quantification. The entire workflow can be completed within 2-4 weeks, from initial design through validation and the production of measurements suitable for cross-study comparisons. Overall, these protocols establish a flexible approach to standardizing organelle quantification so as to understand multiple organelles simultaneously in their cellular contexts. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Mitochondrial quantification Basic Protocol 2: Lipid droplet identification and image processing Basic Protocol 3: Myofibril quantification Basic Protocol 4: Golgi apparatus morphometry Basic Protocol 5: Endoplasmic reticulum network analysis Alternate Protocol: Super-resolution imaging protocol.

Optoelectronic computing devices capable of bipolar responses offer a route to simplified architectures for processing complex tasks. However, advancing such systems towards large-scale, in-sensor computing has been constrained by the difficulty of monolithically integrating neuromorphic optoelectronic arrays with peripheral circuits, largely due to high material growth temperatures and the non-uniform performance of complex device stacks. Here we report Mo4/3B2Tz (Tz = O, OH, F) boridene as a low-thermal-budget platform for neuromorphic optoelectronics, enabling twelve-inch deposition below 150 °C with excellent wafer-scale uniformity. Ordered metal vacancies and interlayer registry variations generate an unusual electrical anisotropy in which through-plane conduction dominates over in-plane transport. This anisotropy enables a simplified three-terminal device architecture that exhibits intrinsic bipolar and highly linear programmable photoresponses. Correlative conductive atomic force microscopy and first-principles simulations reveal that vacancy-mediated interlayer charge transfer governs the observed behaviour. We further fabricate a 54 × 54-pixel2 optoelectronic computing array with a 99.48% yield and 16 fully separable states. Using a 3k-pixel system prototype, we demonstrate the diagnosis of ophthalmic disorders. Our work establishes Mo4/3B2Tz boridene as a scalable nanomaterial platform that brings neuromorphic optoelectronic computing closer to practical implementations.

Cryo-electron tomography (cryo-ET) is a transformative technique in cell biology that enables three-dimensional visualization of cellular structures in near-native states and at nanometer and even subnanometer resolution. Unlike traditional imaging methods, cryo-ET preserves the ultrastructure of cells without chemical fixation or staining, allowing researchers to observe macromolecular complexes in situ. Cryo-focused ion beam milling has overcome sample thickness limitations, enabling high-resolution imaging of complex and large specimens. When combined with correlative light microscopy and subtomogram averaging, cryo-ET can localize and resolve macromolecular assemblies within the cell. We discuss how cryo-ET has provided unprecedented insights into cellular architecture by bridging the gap between molecular and cellular scales and highlight examples in photosynthetic organisms. We also discuss new efforts to increase automation, throughput, and validation that make cryo-ET accessible to a larger community of scientists, including plant biologists.

The joining of dissimilar materials, represented by composites and aluminum alloys, is one of the pathways to achieve structural lightweighting. Preload torque affects the mechanical performance of the structure, making the study of failure evolution behavior in quasi-static tensile loading of carbon fiber reinforced polymer (CFRP) and aluminum alloy bolted connections crucial. This study investigates the effect of preload torque (4 N·m, 8 N·m, and 12 N·m) on the quasi-static tensile evolution of single-lap CFRP-6082-T6 aluminum alloy bolted joints. Through quasi-static tensile tests combined with digital image correlation (DIC) and scanning electron microscopy (SEM), the joint performance was systematically analyzed. Results show that increasing preload torque enhances material stiffness: peak load rises from 10.94 kN to 11.51 kN, and initial damage load increases from 5.17 kN to 8.86 kN. DIC analysis reveals that lower preload torque exacerbates axial compressive strain concentration below the bolt hole and transverse strain concentration at the hole edge, increasing out-of-plane displacement. Higher preload torque mitigates these effects by improving static friction and expanding bolt-hole contact area. Failure modes consistently exhibit net tension failure in CFRP laminates, with cracks initiating at the bolt hole edge and propagating perpendicular to the tensile direction. This study confirms that appropriate preload torque enhances load-bearing capacity and delays initial damage, providing theoretical and experimental support for optimal design of heterogeneous material bolted joints.

Correlative microscopy techniques are used for many different applications in the biological sciences because the comparison of different imaging methods allows researchers to gain more insight and data from samples. Correlative light and electron microscopy (CLEM) methods have been developed to preserve biological samples to withstand the harsh environments necessary for electron microscopy. After first being imaged using widefield (WF) and super-resolution structured illumination fluorescence microscopy (SIM), a NanoSuit chemical treatment was applied to a mammalian testis sample before imaging with scanning electron microscopy (SEM). This was done to compare the image quality and resolution of each technique. SEM yields higher resolution and offers validation of results from SIM.

ABSTRACT Accurate and efficient image segmentation is crucial in anatomy, histology, and pathology research. Conventional manual approaches are time‐consuming, whereas fully automated artificial intelligence segmentation requires substantial manual correction owing to inaccuracy. To address this, we developed SegRef3D, a tool integrating the Segment Anything Model 2 with multiframe tracking and interactive refinement functions, enabling streamlined segmentation workflows for anatomical research. SegRef3D is implemented as a standalone, offline desktop application that operates entirely in a local environment, eliminating the need for cloud‐based services. SegRef3D provides a unified workflow from data import to segmentation, object tracking, refinement, and three‐dimensional model export. Users can specify segmentation prompts through bounding box input, track objects across multiple frames with start–end range selection, and refine results using intuitive Add to Mask and Erase from Mask tools. Up to 20 objects can be handled simultaneously, with each assigned a unique color. The software supports the Standard Tessellation Language output for three‐dimensional modeling and includes volume measurement functions. The SegRef3D prototype, called Seg&Ref, has been applied in studies using serial histological sections, correlative microscopy with block‐face imaging, and pelvic magnetic resonance imaging. Building on these applications, SegRef3D further enhances usability and enables a seamless workflow. SegRef3D offers an accessible, efficient, and accurate segmentation environment tailored for morphological and anatomical studies. Combining artificial intelligence‐powered automatic segmentation with human‐guided refinement in a user‐friendly graphical user interface bridges the gap between research needs and computational methods. By supporting applications that span traditional anatomy and modern pathology, SegRef3D provides a versatile platform for integrative morphological analysis. Its open‐source availability ensures its broad applicability in research, education, and clinical training in the anatomical sciences.

Small extracellular vesicles (sEVs) derived from cytotoxic T lymphocytes (CTLs) are emerging as potential mediators of antitumor immunity; however, their subcellular origins and functional properties remain incompletely defined. In this study, we investigated the intracellular routes and cytotoxic potential of CTL-derived exosomes. Using correlative light and electron microscopy, we discovered that CTL-derived exosomes originate from both classical multivesicular bodies (MVBs) and the recently identified multi core granules (MCGs). Through total internal reflection fluorescence microscopy, we demonstrated that, in contrast to MVB-derived exosomes, MCG-derived exosomes are released at the immunological synapse in a stimulus-dependent manner. To enable functional characterization, we developed a scalable primary cell culture method for the isolation of high-purity exosomes. Super-resolution microscopy revealed significant heterogeneity in exosome size and tetraspanin composition. Notably, MCG-derived exosomes exhibited fivefold higher cytotoxic activity than MVB-derived exosomes, inducing apoptosis in tumor cells via a caspase 3-dependent mechanism. These findings reveal that CTLs exploit distinct secretory pathways to release heterogeneous exosome populations with differential cytotoxic capacities, offering new insights into CTL-mediated immune responses and providing a basis for the development of novel exosome-based immunotherapies.

ABSTRACT Safe and permanent storage of CO 2 via Carbon Capture and Storage (CCS) technologies is required to limit global warming to 1.5°C–2°C above pre‐industrial levels. In situ mineralization of CO 2 within reactive formations or silicate‐rich materials containing Ca‐ and Mg‐bearing minerals, such as cement and basalt, is a potentially rapid and secure method of geological CO 2 storage. We review how advanced imaging techniques including X‐ray microcomputed tomography and electron microscopy can be integrated to understand the CO 2 mineralization process at the microscale. We highlight how novel methods, including correlative microscopy and machine learning, are pivotal in studying complex reactions within heterogeneous samples. We explain how integrating microstructure analysis increases the reliability of reactive transport modeling and consequently improves the CO 2 storage capacity assessment. Furthermore, we discuss challenges that need addressing to improve accuracy in measuring properties like reactive surface area and highlight the key areas for future research, including method validation and error estimation of properties acquired through imaging. While image analysis offers profound insights into CO 2 mineralization, significant advancement is required in segmentation accuracy and reproducibility. Furthermore, using experimental observations from studies of cement and basalt, we suggest a decision tree for assessing the suitability of formations for CO 2 mineralization.

The ability to track individual centrioles in living cells represents a pivotal methodology for advancing our understanding of centrosome biology, cell division, and ciliogenesis. This article provides a comprehensive review of the current methodological landscape for centriole tracking, bridging historical approaches with cutting-edge innovations. We begin by outlining the fundamental challenges—including the organelle's sub-diffraction size, dynamic life cycle, and close pairing—that have historically limited observation. The review then details a hierarchical progression of techniques, from foundational static methods like immunofluorescence and electron microscopy to the direct live-cell imaging enabled by fluorescent protein fusions and advanced microscopy platforms such as spinning-disk confocal and Total Internal Reflection Fluorescence (TIRF) microscopy. A significant focus is placed on super-resolution methods, particularly Stimulated Emission Depletion (STED) microscopy, which allows for the resolution of individual centrioles within a pair in real time. We further explore critical genetic manipulation and labeling strategies, including CRISPR/Cas9-mediated endogenous tagging and photoactivatable proteins for pulse-chase experiments. Practical, detailed protocols for long-term tracking, super-resolution imaging, and lineage analysis are presented, followed by an in-depth discussion of the computational pipeline for data analysis, encompassing object detection, trajectory linking, and quantitative kinetic measurements. Finally, we address common artifacts and mitigation strategies, and conclude by highlighting emerging technologies like correlative light-electron microscopy (CLEM) and lattice light-sheet microscopy (LLSM) that promise to further revolutionize the field by linking dynamic behavior with ultrastructure and enabling studies within complex 3D tissues.

Visualization of neuronal ultrastructure facilitates molecular and biochemical analyses that may help to better elucidate neural function and information processing. While the neuron exists at the micron scale, critical events such as synaptic vesicle release and dendritic spine remodeling occur at the nanometer scale, necessitating submicron resolution. Scanning electron microscopy (SEM) provides high-resolution imaging at these scales. However, the commonly used dehydration-based sample preparation method induces morphological distortions, while environmental SEM requires specialized equipment that is costly and difficult to operate. The NanoSuit method has recently emerged as a promising alternative, enabling SEM observations under high-vacuum conditions without standard (dehydration-based) pretreatment. Although known to be successful when applied to specimens with protective surface layers such as insects, flowers, and wet tissues, its effectiveness when examining "bare" cultured cells has not been thoroughly explored. Here, we present a modified NanoSuit protocol for SEM examination of cultured neurons and compare it with standard pretreatment. We demonstrate that traditional methods frequently cause neuronal transection and loss of fine dendritic processes, particularly during early development of neurons. However, the modified NanoSuit approach preserves neuronal morphology, enabling clear visualization of thin neurites and their interactions. Further, we successfully implemented correlative light and electron microscopy (CLEM) using this method, enabling the colocalization of cytoskeletal proteins such as actin and tubulin with the surface features observed by SEM. This combination of morphological preservation and molecular localization provides a more accurate and holistic understanding of neuronal structures, benefiting studies on neural development, synaptic connectivity, and related biomedical applications.

Homoepitaxial growth of highly crystalline microribbons on large-area 2D transition metal dichalcogenide (TMD) monolayers is often facilitated by molten salt chemical vapor deposition (CVD) via the vapor-liquid-solid growth method. These microribbons are vertically stacked TMD monolayer stripesthat exhibit well-defined edge structures, introducing spatial heterogeneities across the basal plane of their 2D monolayer supports. Although frequently observed in CVD-grown TMDs, their influence on local electrocatalytic behavior remains poorly understood. Here, we use scanning electrochemical cell microscopy (SECCM), combined with correlative microscopy and spectroscopy, to spatially resolve the hydrogen evolution reaction (HER) activity of WS2 monolayer decorated with microribbons at nanometer resolution. Quantitative analysis of over 33000 localized electrochemical measurements reveals that monolayer edges exhibit twice the catalytic activity of the basal plane, while single- and multilayer microribbons suppress HER activity by over 5- and 10-fold, respectively, due to interlayer coupling and sluggish charge transport from the underlying Au substrate. Overall, this work reveals how microribbons modulate basal plane activity in 2D WS2 on metallic Au substrate and demonstrates how co-localization of SECCM with various spectroscopies can be used as a powerful technique for mapping nanoscale catalytic activity across structurally complex TMD surfaces.

The autophagy receptor p62 is degraded via autophagy under hyperosmotic stress, but whether this involves the formation of biomolecular condensates (p62 bodies) remains unclear. Using human cells, we found that p62 bodies formed within 1 minute of hyperosmotic stress, and increased with stress severity. They formed faster and under milder stress than stress granules, a classic condensate, and exhibited liquid-like properties. Unlike stress granules, p62 bodies frequently colocalized with LC3 and WIPI-2, and were degraded via autophagy. Correlative light and electron microscopy revealed that these p62 bodies were more compact than stress granules and were often associated with the autophagic isolation membrane. Autophagy receptors NBR1 and TAX1BP1, but not OPTN1 or NDP52, behaved similarly to p62, and p62 bodies preferentially contained K63-linked ubiquitin chains. p62 body formation was also observed in human epithelial organoids in association with WIPI-2. Collectively, these results indicate that p62 bodies function as a platform of degradation under hyperosmotic stress.

ABSTRACT The quasi‐static shear mechanical behavior and damage mechanisms of high‐silica woven fiber‐reinforced phenolic interfaces after thermal‐oxidative aging and fatigue were investigated through shear tests in conjunction with digital image correlation (DIC) and scanning electron microscopy (SEM). The residual shear strength of interface progressively decreases with increasing fatigue amplitude, cycles, and duration of thermal‐oxidative aging. Up to 20,000 cycles, the residual strengths under fatigue loadings , , , and decreased by 8.9%, 12.9%, 21.3%, and 30.3%, respectively. At fatigue amplitude , thermo‐oxidative aging for 36, 76, and 103 days caused reductions of 30.4%, 34.6%, and 38.4% relative to the unaged specimens. DIC and SEM reveal that interfacial shear damage initiates at defects along the biphasic interface, progresses through microcrack propagation with concurrent penetration into the interlaminar region, and ultimately results in crack coalescence and macroscopic delamination‐induced instability failure. Under fatigue‐dominated loading, damage is characterized by fiber fracture and interlaminar debonding, whereas thermo‐oxidative aging suppresses fiber tearing but markedly exacerbates interlaminar debonding. Based on the equivalence principle of residual strength under fatigue loading‐cycling‐aging, a predictive model for residual shear strength was developed to account for the effects of thermal‐oxidative aging and fatigue. Experimental validation showed that the model achieved an average relative error of approximately 10%, demonstrating good agreement with the measured data.

• Correlative microscopy reveals laser-induced surface modifications in WC-Co. • Laser treatment accentuates pre-existing defects from material preparation. • Oxidation is non-uniform, with higher levels on certain flanks of laser-ablated microgrooves. • Nanomechanical properties correlate with local chemical composition and topography. Comprehensive correlative characterization of material surfaces is essential for understanding how certain treatments, such as laser treatments, can modify their properties and overall performance. This study employs a multi-modal correlative microscopy approach, combining Scanning Electron Microscopy (SEM), Energy Dispersive X-ray Spectroscopy (EDX), Optical Microscopy (OM), Focus-Variation Optical Microscopy (F-V OM) and Atomic Force Microscopy (AFM) to characterize laser-textured Tungsten Carbide-Cobalt (WC-Co) surfaces. The characterization was carried out on the same area before and after pulsed laser treatment at ambient atmosphere. The experiments revealed the persistence of surface defects, presumably caused by Wire Electrical Discharge Machining (WEDM) during sample preparation, which were further accentuated by the subsequent laser treatment. In addition, significant and non-uniform surface oxidation was observed, with elevated levels on specific flanks of laser-ablated grooves. This analysis demonstrated that spatial property decoupling is key, as the maximum topographic height, nearing 7 µm height from the lowest surface point, did not spatially coincide with the extremes of the chemical composition or total deformation curves. Specifically, point analysis showed that oxygen concentrations varied sharply, reaching 29.02 atomic oxygen percentage on high-deformation zones, contrasting with 21.18 at.% O found at adjacent low-deformation zones. Finally, the relationship among topography, chemical composition and nanomechanical properties was demonstrated, and the value of applying multi-modal correlative microscopy for understanding laser-material interactions was underscored. The insights gained highlight the potential of the methodology for optimizing laser parameters to achieve targeted surface functionalities.

Coordination of N1 and N2-methylated regioisomers of 2-(1H-tetrazol-5-yl)pyridine and 2-(1H-tetrazol-5-yl)quinoline to ReBr(CO)3 and Ir(ppy)2+ (ppy = cyclometalated 2-phenylpyridine) fragments resulted in the isolation of a small family of Re(I) and Ir(III) luminescent complexes. The complexes display phosphorescent emission from their triplet ligand-to-metal charge transfer excited states in degassed solution at room temperature. Notably, the position of the methyl substituent has a profound effect on the photophysical properties. The N1-methylated complexes in all cases display a significant redshift in the emission band. The shift is ascribed to a stabilisation of the π* orbitals of the tetrazole ligands, which is also supported by cyclic voltammetry and time-dependent density functional theory (TD-DFT) calculations. The complexes were used as luminescent labels for the staining of mouse brain tissue. The Re(I) complexes did not show any evident staining. On the other hand, the Ir(III) complexes - particularly those bound to the ligand containing the quinoline substituent - demonstrated affinity for lipid-rich myelinated regions in brain tissues and white matter in cerebellum tissues. The specificity of the Ir(III) complexes was further demonstrated by means of correlative optical microscopy and Fourier transform infrared (FTIR) microscopy.