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A three‐dimensional (3D) microstructural volume is reconstructed from a stack of two‐dimensional sections which was obtained by serial sectioning coupled with electron back scattering diffraction (EBSD) mapping of a 316L austenitic stainless steel. A new alignment algorithm named linear translation by minimising the indicator (LTMI) is proposed to reduce the translational misalignments between adjacent sections by referencing to coherent twin boundaries which are flat and lying on {111} planes. The angular difference between the measured orientation of a flat twin boundary and that of the {111} plane is used as an indicator of the accuracy of the alignment operations. This indicator is minimised through linear translations of the centroids of triangular facets, which constitute grain boundaries at a distance not restricted by the in‐plane step size of the EBSD maps. And hence the systematic trend in the translational misalignments can be effectively reduced. The LTMI alignment procedure proposed herein effectively corrects the misalignments remained by other methods on a 3D‐EBSD data prepared using serial sectioning methods. The accuracy in distinguishing between coherent and incoherent twin boundaries is significantly improved. LAY DESCRIPTION: Accurately reconstructing the three‐dimensional (3D) microscopic structure of metal materials is crucial for exploring the relationship between the structure and properties of materials. Researchers utilise electron backscatter diffraction (EBSD) technique to sequentially acquire two‐dimensional (2D) images layer by layer, which are then stacked to reconstruct the three‐dimensional (3D) microstructure of the material. However, misalignments during this stacking process can result in an incorrect alignment between the layers, leading to distortion of the entire 3D volume. Previous methods aligned layers by making adjacent layers as similar as possible, but this could introduce cumulative errors. This work has proposed a new alignment method, 'Linear translation by minimising the indicator' (LTMI), which utilises coherent twin boundaries as additional references to guide the stacking process and accurately find the correct position between layers. Coherent twin boundaries serve as references because of their characteristics: these boundaries are special structures within the material, having a flat morphology and lying on specific crystal planes. Layers are shifted repeatedly until all the coherent twin boundaries in the sample display these characteristics, signifying that the layers are properly aligned. The LTMI method offers a way to significantly enhance the accuracy of 3D microstructural analysis, which is essential for designing materials that are strong and durable, with profound implications for the materials field. [ABSTRACT FROM AUTHOR]

This paper describes the development of a three-dimensional (3D) indentation test system capable of observing the distribution of mechanical properties in structural materials. Serial sectioning with destructive treatment has traditionally been used as a method for observing microstructure within materials in three dimensions. The serial sectioning methods using precision cutting has attracted particular attention as it enables the observation of large sample volumes. However, those methods can only observe the microstructure as image, not the mechanical properties such as hardness and elastic modulus. To measure the 3D distribution of the mechanical properties of the material, it is effective to combine repeated cutting and indentation tests on each cutting surface. Morever, combining the image observation and mechanical property tests could allow a more sophisticated analysis of the interior of material. To implement this method, we have constructed an indentation test system on a precision machine using a Berkovich indenter, micro-force sensor, and micro-movement stage. In order to achieve a 3D indentation test, it is considered necessary to unify the measurement positions in the depth direction. Furthermore, the unloading rate needs to be controlled in order to carry out stable indentation tests. Therefore, we propose a method of 3D indentation test that can precisely control the maximum depth of indentation and unloading speed. In this paper, we devise a method for driving the constructed system and a method for obtaining data and confirm the accuracy of these methods by experiment. In addition, we determine indentation depth and unloading speed which are suitable for our method by performing indentation tests on a block for ultra-microhardness. Finally, we practice 3D indentation test in which the cutting and indentation tests are repeated on specimens with different mechanical properties in the depth direction. Experimental results show that our indentation test system is appropriate to measure three-dimensional mechanical properties inside the material. • The system was devised to enable indentation test on the precision machining. • The system allowed repeatable indentation tests on the cutting surface. • The indentation test system was built using a piezoelectric stage and force sensor. • This indentation test system could control indentation depth and unloading speed. [ABSTRACT FROM AUTHOR]

We discovered that the light entering a triangular ultramicrotome glass knife from the bottom exits the knife through its cutting edge, forming an oblique light sheet illumination suitable for imaging. We adopted this light sheet for side illumination of the sample blocks during sectioning on the ultramicrotome, for 3D imaging, and for targeting fluorescent features for confocal‐, electron‐ and correlative microscopy. In this paper, we present a working prototype named CELS‐3D (Cutting Edge Light Source, Three‐Dimensional), a microscope mounted on an ultramicrotome. We characterised CELS‐3D and applied it for 3D imaging of human liver spheroids with a diameter of approximately 500 μm. The structure of nuclei and tight junctions has been successfully reconstructed over the full spheroid volume. In contrast, a confocal microscope was unable to image spheroids to a depth of greater than 50 μm. CELS‐3D shows fluorescence during serial sectioning in an online mode; therefore, it can apply for targeting fluorescence structures for correlative microscopy. We successfully applied CELS‐3D for targeted correlative microscopy of human liver spheroids and C. elegans. The CELS‐3D can be utilised for less‐ and non‐transparent samples, which encompasses a range of applications, including operation biopsies, experimental organoids/spheroids, artificial cartilage, and bone, among others. The CELS‐3D can be effortlessly mounted on the top of any commercially available ultramicrotome, and its operation is straightforward and intuitive. [ABSTRACT FROM AUTHOR]

Tissue slicing is at the core of many approaches to studying biological structures. Among the modern volume electron microscopy (vEM) methods, array tomography (AT) is based on serial ultramicrotomy, section collection onto solid support, imaging via light and/or scanning electron microscopy, and re-assembly of the serial images into a volume for analysis. While AT largely uses standard EM equipment, it provides several advantages, including long-term preservation of the sample and compatibility with multi-scale and multi-modal imaging. Furthermore, the collection of serial ultrathin sections improves axial resolution and provides access for molecular labeling, which is beneficial for light microscopy and immunolabeling, and facilitates correlation with EM. Despite these benefits, AT techniques are underrepresented in imaging facilities and labs, due to their perceived difficulty and lack of training opportunities. Here we point towards novel developments in serial sectioning and image analysis that facilitate the AT pipeline, and solutions to overcome constraints. Because no single vEM technique can serve all needs regarding field of view and resolution, we sketch a decision tree to aid researchers in navigating the plethora of options available. Lastly, we elaborate on the unexplored potential of AT approaches to add valuable insight in diverse biological fields. [ABSTRACT FROM AUTHOR]

Here, we summarize the literature relevant to recent advances in three‐dimensional (3D) histopathology in relation to clinical oncology, highlighting serial sectioning, tissue clearing, light‐sheet microscopy, and digital image analysis with artificial intelligence. We look forward to a future where 3D histopathology expands our understanding of human pathophysiology and improves patient care through cross‐disciplinary collaboration and innovation. [ABSTRACT FROM AUTHOR]

X-ray computed tomography (XCT) is a widely adopted nondestructive technique for characterizing internal porosity in additive manufactured (AM) components. However, the accuracy and precision of porosity characterization using XCT can be affected by factors, such as XCT system configuration and post-processing methodologies. This study investigates the influence of these variables on porosity characterization by comparing results obtained from four different XCT systems and two distinct analysis workflows applied to a single metallic AM sample. A benchmark is also established for the XCT performance by using a high-resolution reference dataset generated through mechanical polishing serial sectioning (MPSS). Porosity metrics, including volume fraction, pore count, size distribution, and equivalent spherical diameter (ESD), were computed for large pores ( $\ge 84~\mu $ m) within the XCT and MPSS datasets. By comparing these metrics across XCT systems and workflows, this research aims to demonstrate the variability introduced by different XCT configurations and analysis procedures, providing insights into the potential limitations and uncertainty considerations needed while carrying out XCT-based porosity characterization of AM components.

Isotopic, tagging and diet studies of modern‐day teleosts lacked the ability to contextualise life‐history and trophic dynamics with a historical perspective, when exploitation rates were lower and climatic conditions differed. Isotopic analysis of vertebrae, the most plentiful hard‐part in archaeological and museum collections, can potentially fill this data‐gap. Chemical signatures of habitat and diet use during growth are retained by vertebrae during bone formation. Nonetheless, to fulfil their potential to reveal life‐history and trophic dynamics, we need a better understanding of the time frame recorded by vertebrae, currently lacking due to a poor understanding of fish bone remodelling. To address this issue, the authors serially‐sectioned four vertebral centra of the highly migratory Atlantic bluefin tuna (Thunnus thynnus; BFT) captured off Sardinia (Italy) and analysed their isotopic composition. They show how carbon (δ13C), nitrogen (δ15N) and sulphur (δ34S) isotope values can vary significantly across BFT vertebrae growth‐axes, revealing patterning in dietary life histories. Further, they find that similar patterns are revealed through incremental isotopic analysis of inner and outer vertebrae centra samples from 13 archaeological BFT vertebrae dating between the 9th and13th centuries CE. The results indicate that multi‐year foraging signatures are retained in vertebrae and allow for the study of life histories in both modern and paleo‐environments. These novel methods can be extended across teleost taxa owing to their potential to inform management and conservation on how teleost trophic dynamics change over time and what their long‐term environmental, ecological and anthropological drivers are. [ABSTRACT FROM AUTHOR]

Tundra shrubs reflect climate sensitivities in their growth-ring widths, yet tissue-specific shrub chronologies are poorly studied. Further, the relative importance of regional climate patterns that exert mesoscale precipitation and temperature influences on tundra shrub growth has been explored in only a few Arctic locations. Here, we investigate Betula nana growth-ring chronologies from adjacent dry heath and moist tussock tundra habitats in arctic Alaska in relation to local and regional climate. Mean shrub and five tissue-specific ring width chronologies were analyzed using serial sectioning of above- and below-ground shrub organs, resulting in 30 shrubs per site with 161 and 104 cross sections from dry and moist tundra, respectively. Betula nana growth-ring widths in both habitats were primarily related to June air temperature (1989–2014). The strongest relationships with air temperature were found for 'Branch2' chronologies (dry site: r = 0.78, June 16, DOY = 167; moist site: r = 0.75, June 9, DOY = 160). Additionally, below-ground chronologies ('Root' and 'Root2') from the moist site were positively correlated with daily mean air temperatures in the previous late-June ('Root2' chronology: r = 0.57, pDOY = 173). Most tissue-specific chronologies exhibited the strongest correlations with daily mean air temperature during the period between 8 and 20 June. Structural equation modeling indicated that shrub growth is indirectly linked to regional Arctic and Pacific Decadal Oscillation (AO and PDO) climate indices through their relation to summer sea ice extent and air temperature. Strong dependence of Betula nana growth on early growing season temperature indicates a highly coordinated allocation of resources to tissue growth, which might increase its competitive advantage over other shrub species under a rapidly changing Arctic climate. [ABSTRACT FROM AUTHOR]

Objective: Serial sectioning optical coherence tomography (OCT) enables accurate volumetric reconstruction of several cubic centimeters of human brain samples. We aimed to identify anatomical features of the ex vivo human brain, such as intraparenchymal blood vessels and axonal fiber bundles, from the OCT data in 3D, using intrinsic optical contrast. Methods: We developed an automatic processing pipeline to enable characterization of the intraparenchymal microvascular network in human brain samples. Results: We demonstrated the automatic extraction of the vessels down to a 20 ${\boldsymbol{\mu }}$ m in diameter using a filtering strategy followed by a graphing representation and characterization of the geometrical properties of microvascular network in 3D. We also showed the ability to extend this processing strategy to extract axonal fiber bundles from the volumetric OCT image. Conclusion: This method provides a viable tool for quantitative characterization of volumetric microvascular network as well as the axonal bundle properties in normal and pathological tissues of the ex vivo human brain. [ABSTRACT FROM AUTHOR]

Mosaic analysis with double markers (MADM) is a powerful in vivo lineage tracing technique. It utilizes Cre recombinase-dependent interchromosomal recombination to restore the stable expression of two fluorescent proteins sparsely in individual dividing stem or progenitor cells and their progenies. Here, we describe the application of this technique for quantitative lineage analysis of radial glial progenitors in the developing mouse neocortex at the single-cell resolution., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Accelerating Arctic permafrost thaw results in increasing methane emissions affecting regional and global climates, but the rate of permafrost disappearance can be difficult to estimate with remote sensing, field surveys and modelling. Here we investigate if the annual growth rings of the shrub Betula nana (dwarf birch) may be used to detect and monitor near-surface permafrost degradation. Whole B. nana samples were collected at Latnjajávri and Corrvosjávri, northernmost Sweden, and their annual growth rings were analysed regarding their potential as permafrost indicators. Permafrost disappeared in Latnjajávri between 1993 and 2001, whereas Corrvosjávri lost its frozen ground decades earlier. Annual growth rings from more than twenty B. nana shrubs at each site were measured and crossdated by serial sectioning, and then averaged into two separate shrub-ring chronologies. Growth rates were higher at Corrvosjávri than at Latnjajávri. Analysing the 1972–2015 period disclosed a stronger climate-growth relationship at Corrvosjávri, with significant correlations with summer temperatures. In Latnjajávri the association with climate was much weaker before the permafrost disappeared, but afterwards reached similar levels as at Corrvosjávri. Our results suggest that changes in the B. nana growth-climate relationship may be used to indicate permafrost degradation. Moreover, the strong correlation between summer temperature and shrub growth after disappearance of permafrost supports previous research that B. nana ring widths are useful climate proxies. [ABSTRACT FROM AUTHOR]

Chemical mapping by Raman spectroscopy is widely used in the pharmaceutical industry to characterise the distribution of components within pharmaceutical tablets; however, current methods do not go beyond examining an exposed surface area of a sample. There are known limitations with estimating domain size and shape statistics from 2D chemical images as the values obtained will depend on where the domain is sectioned, potentially under‐ or overestimating its true value. The combination of Raman spectroscopic mapping and serial sectioning has been recently explored as an alternative method to obtain a depth profile of a sample; however, to date, this has involved instrumentation capable of automated Raman mapping with subsequent sample sectioning. A key requirement for Raman mapping is producing an optically flat surface, and this becomes increasingly challenging for larger surface areas required for the examination of a pharmaceutical tablet. Here, we describe 3D imaging of a tablet matrix by combining Raman mapping with independent sample sectioning to provide appropriate lateral and axial resolution. The approach was first validated by analysing a spherical object of known size and shape and comparing the 3D domain size statistics calculated from the reconstructed image to its absolute values. The method was then applied to a three‐component model system, simulating a pharmaceutical tablet, to determine the capability and applicability of the method for solid dosage formulations. The study demonstrated that relative differences in the size, shape and distribution of domains can be quantified enabling an enhanced understanding of the spatial arrangement of each component within the formulation and the effect of each processing condition on the final drug product. By visualising the 3D structure of a tablet matrix with demonstrable accuracy and precision using materials of known dimensions, new capabilities to enhance tablet manufacturing methods are now available. [ABSTRACT FROM AUTHOR]

This paper describes a fast and precise 3D observation method for steel microstructures using a cutting-based serial sectioning system with on-site etching. The method employs automated 3D internal structure microscopy for ferrous materials and on-site manual etching without detaching the specimen during the process. A notable feature of this system is that it uses precision cutting instead of polishing for surface fabrication, which has advantages in the fabrication speed, applicability in dry conditions, and controllability of the fabrication depth. The precision cutting of a dual-phase (DP) steel achieved a low roughness comparable to polishing. Furthermore, the on-site etching process by dropping etchant yielded sufficient contrast between ferrite and martensite phases. Observation of DP steel cross sections at 1 μm intervals afforded a 3D image in the range of 690 × 518 × 103 μm3 with a resolution of 0.144 × 0.144 × 1.0 μm3. The processing time was approximately five minutes for each cross section and almost half a day for the whole image, which was much more efficient than manual serial sectioning that requires a few weeks. The microstructures were also visible in reconstructed lateral cross sections of the 3D image, which proved the sufficient quality of our imaging. In addition, the segmented volume revealed the non-uniform martensite distribution among cross sections quantitatively. Most of them were connected in 3D, while they seemed to be separated in 2D. These results demonstrated the usefulness of our method with precision cutting and on-site etching. [Display omitted] • Cutting-based sectioning and manual etching for 3D steel microstructure observation. • Low roughness and sufficient contrast between ferrite and martensite phases. • High resolution and fabrication time (5 min per cross section; half day per image). • Microstructures observation in reconstructed lateral cross sections. • Observation of non-uniform martensite distribution among cross sections. [ABSTRACT FROM AUTHOR]

The structural complexity of nervous tissue makes it very difficult to unravel the connectivity between neural elements at different scales. Numerous methods are available to trace long-range projections at the light microscopic level, and to identify the actual synaptic connections at the electron microscopic level. However, correlating mesoscopic and nanoscopic scales in the same cell, cell population or brain region is a problematic, laborious and technically demanding task. Here we present an effective method for the 3D reconstruction of labeled subcellular structures at the ultrastructural level, after single-neuron labeling in fixed tissue. The brain is fixed by intracardial perfusion of aldehydes and thick vibratome sections (250 μm) are obtained. Single cells in these vibratome sections are intracellularly injected with horseradish peroxidase (HRP), so that the cell body and its processes can be identified. The thick sections are later flat-embedded in epoxy resin and re-sectioned into a series of thinner (7 μm) sections. The sections containing the regions of interest of the labeled cells are then imaged with automated focused ion beam milling and scanning electron microscopy (FIB-SEM), acquiring long series of high-resolution images that can be reconstructed, visualized, and analyzed in 3D. With this methodology, we can accurately select any cellular segment at the light microscopic level (e.g., proximal, intermediate or distal dendrites, collateral branches, axonal segments, etc.) and analyze its synaptic connections at the electron microscopic level, along with other ultrastructural features. Thus, this method not only facilitates the mapping of the synaptic connectivity of single-labeled neurons, but also the analysis of the surrounding neuropil. Since the labeled processes can be located at different layers or subregions, this method can also be used to obtain data on the differences in local synaptic organization that may exist at different portions of the labeled neurons. [ABSTRACT FROM AUTHOR]

2D sections of rice (Oryza sativa L.) mesophyll cells are complex and the cell interior contains a large volume of chloroplasts. Furthermore, the structure of chloroplasts changes in response to salt stress. In this study, we used a 3D reconstruction method based on serial section light microscopy to analyze a wide range of structures in leaf tissues and compared the intracellular structure of mesophyll cells of rice under normal and salt-treated conditions. The 3D reconstructed models revealed that mesophyll cells appeared as ellipsoid discs with several lobes around the cell periphery, with the volumes showing no significant difference between control and salt-treated plants. The chloroplast structure in the whole mesophyll cell was altered under salt stress, showing a reduced coverage area, whereas their volumes did not differ between treatments. These findings suggest that 3D reconstruction based on serial light micrographs can reveal the morphology of cells and chloroplasts in plant tissue.

The structural complexity of nervous tissue makes it very difficult to unravel the connectivity between neural elements at different scales. Numerous methods are available to trace long-range projections at the light microscopic level, and to identify the actual synaptic connections at the electron microscopic level. However, correlating mesoscopic and nanoscopic scales in the same cell, cell population or brain region is a problematic, laborious and technically demanding task. Here we present an effective method for the 3D reconstruction of labeled subcellular structures at the ultrastructural level, after single-neuron labeling in fixed tissue. The brain is fixed by intracardial perfusion of aldehydes and thick vibratome sections (250 μm) are obtained. Single cells in these vibratome sections are intracellularly injected with horseradish peroxidase (HRP), so that the cell body and its processes can be identified. The thick sections are later flat-embedded in epoxy resin and re-sectioned into a series of thinner (7 μm) sections. The sections containing the regions of interest of the labeled cells are then imaged with automated focused ion beam milling and scanning electron microscopy (FIB-SEM), acquiring long series of high-resolution images that can be reconstructed, visualized, and analyzed in 3D. With this methodology, we can accurately select any cellular segment at the light microscopic level (e.g., proximal, intermediate or distal dendrites, collateral branches, axonal segments, etc.) and analyze its synaptic connections at the electron microscopic level, along with other ultrastructural features. Thus, this method not only facilitates the mapping of the synaptic connectivity of single-labeled neurons, but also the analysis of the surrounding neuropil. Since the labeled processes can be located at different layers or subregions, this method can also be used to obtain data on the differences in local synaptic organization that may exist at different portions of the labeled neurons.

We present a software‐assisted workflow for the alignment and matching of filamentous structures across a three‐dimensional (3D) stack of serial images. This is achieved by combining automatic methods, visual validation, and interactive correction. After the computation of an initial automatic matching, the user can continuously improve the result by interactively correcting landmarks or matches of filaments. Supported by a visual quality assessment of regions that have been already inspected, this allows a trade‐off between quality and manual labour. The software tool was developed in an interdisciplinary collaboration between computer scientists and cell biologists to investigate cell division by quantitative 3D analysis of microtubules (MTs) in both mitotic and meiotic spindles. For this, each spindle is cut into a series of semi‐thick physical sections, of which electron tomograms are acquired. The serial tomograms are then stitched and non‐rigidly aligned to allow tracing and connecting of MTs across tomogram boundaries. In practice, automatic stitching alone provides only an incomplete solution, because large physical distortions and a low signal‐to‐noise ratio often cause experimental difficulties. To derive 3D models of spindles despite dealing with imperfect data related to sample preparation and subsequent data collection, semi‐automatic validation and correction is required to remove stitching mistakes. However, due to the large number of MTs in spindles (up to 30k) and their resulting dense spatial arrangement, a naive inspection of each MT is too time‐consuming. Furthermore, an interactive visualisation of the full image stack is hampered by the size of the data (up to 100 GB). Here, we present a specialised, interactive, semi‐automatic solution that considers all requirements for large‐scale stitching of filamentous structures in serial‐section image stacks. To the best of our knowledge, it is the only currently available tool which is able to process data of the type and size presented here. The key to our solution is a careful design of the visualisation and interaction tools for each processing step to guarantee real‐time response, and an optimised workflow that efficiently guides the user through datasets. The final solution presented here is the result of an iterative process with tight feedback loops between the involved computer scientists and cell biologists. Lay description: Electron tomography of biological samples is used for a three‐dimensional (3D) reconstruction of filamentous structures, such as microtubules (MTs) in mitotic and meiotic spindles. Large‐scale electron tomography can be applied to increase the reconstructed volume for the visualisation of full spindles. For this, each spindle is cut into a series of semi‐thick physical sections, from which electron tomograms are acquired. The serial tomograms are then stitched and non‐rigidly aligned to allow tracing and connecting of MTs across tomogram boundaries. Previously, we presented fully automatic approaches for this 3D reconstruction pipeline. However, large volumes often suffer from imperfections (ie physical distortions) caused by the image acquisition process, making it difficult to apply fully automatic approaches for matching and stitching of numerous tomograms. Therefore, we developed an interactive, semi‐automatic solution that considers all requirements for large‐scale stitching of microtubules in image stacks of consecutive sections. We achieved this by combining automatic methods, visual validation and interactive error correction, thus allowing the user to continuously improve the result by interactively correcting landmarks or matches of filaments. We present large‐scale reconstructions of spindles in which the automatic workflow failed and where different steps of manual corrections were needed. Our approach is also applicable to other biological samples showing 3D distributions of MTs in a number of different cellular contexts. [ABSTRACT FROM AUTHOR]

High-energy diffraction microscopy (HEDM) in-situ mechanical testing experiments offer unique insight into the evolving deformation state within polycrystalline materials. These experiments rely on a sophisticated analysis of the diffraction data to instantiate a 3D reconstruction of grains and other microstructural features associated with the test volume. For microstructures of engineering alloys that are highly twinned and contain numerous features around the estimated spatial resolution of HEDM reconstructions, the accuracy of the reconstructed microstructure is not known. In this study, we address this uncertainty by characterizing the same HEDM sample volume using destructive serial sectioning (SS) that has higher spatial resolution. The SS experiment was performed on an Inconel 625 alloy sample that had undergone HEDM in-situ mechanical testing to a small amount of plastic strain (~ 0.7%), which was part of the Air Force Research Laboratory Additive Manufacturing (AM) Modeling Series. A custom-built automated multi-modal SS system was used to characterize the entire test volume, with a spatial resolution of approximately 1 µm. Epi-illumination optical microscopy images, backscattered electron images, and electron backscattered diffraction maps were collected on every section. All three data modes were utilized and custom data fusion protocols were developed for 3D reconstruction of the test volume. The grain data were homogenized and downsampled to 2 µm as input for Challenge 4 of the AM Modeling Series, which is available at the Materials Data Facility repository. [ABSTRACT FROM AUTHOR]

Three-dimensional (3D) observations of internal structures are important for evaluating material properties. Serial sectioning with destructive processes is traditionally employed as a 3D observation method. Identifying the boundaries of elements in microscope images and measuring the mechanical properties of each element are required for the evaluation of the mechanical properties of composite materials. This study provides a system for measuring the local hardness and elastic modulus by conducting indentation tests during serial sectioning processes. An automatic serial sectioning observation was performed during a combination process of precision cutting in high-speed milling with a single-crystal diamond tool and microscopic observation. A Vickers indenter was attached to a tool spindle table, and indentation tests were conducted under a displacement control process at submicron spatial resolution. The indentation modulus was obtained by analyzing the force–displacement profile measured during the unload process. The scale effects relating to the indentation depth in the measurements of the indentation modulus were confirmed for an Al alloy sample measured in this system. This study focused on the identification of components by using hardness information measured under the same indentation depth on a two-dimensional flat surface after precision cutting of the material. Three types of metal wires (1 mm diameter) embedded in plastic resin were used in the experiment. The hardness distributions on the serial sectioning surfaces were measured, and the values measured at each wire area on 3D positions were used for the identification of their material properties. This serial sectioning observation creates a 3D microstructural model including not only microscopic images, but also hardness and elastic modulus information for the identification of components in the microscopic area. • Multipoint indentations were automatically controlled in a serial sectioning system. • 3D microstructures of metal wires were constructed by serial sectioning observations. • Material identification was performed using indentation characteristics. [ABSTRACT FROM AUTHOR]

Objectives: The growth of tooth dentin is incremental, so its formation represents a dietary record in early life. With archeological skeletons, applying sequential stable isotope analysis to the horizontal sections of tooth dentin has revealed weaning patterns and dietary changes that took place during childhood. However, the assignment of ages to dentin serial sections (DSSs) is problematic due to the changing extension rate and oblique growth layers of dentin, and these effects have not been quantified. This study presents a mathematical model for investigating the corresponding age range of the horizontal DSSs of human permanent incisors, canines, and molars. Methods: Parameters describing the tooth dentin microstructure were taken from previous studies, and dentin growth patterns were modeled. The model was implemented as the R package MDSS. Results: The developed model shows that the true corresponding age of the sections differed by a few years on average from the estimated age with equal temporal divisions, that the model gave values extending across a wide range, and that these differences become large for sections formed at older ages. The stable isotope ratio of the sections presented an aggregate representation of possibly complex dietary changes across a few years, and dietary changes over short times, such as several months, could not be accurately reconstructed even when using a finer horizontal sectioning method. Conclusions: These results demonstrate that DSSs correspond to an unexpectedly wider (i.e., three to four times) and different (i.e., −2 to 0.5 years on average) age range than previously assumed and that complicated patterns of dietary change blur in the isotopic trajectory of the sections. Alternative experimental methods, such as imaging‐assisted oblique sampling, should be used to retrieve an accurate and precise sequential dietary record from tooth dentin. [ABSTRACT FROM AUTHOR]

Confocal Raman microscopy enables 3D imaging of various samples solely based on chemical contrast. However, optical artifacts impair resolution and image quality in subsurface imaging. With serial section Raman tomography, we show that serial ultrathin and semithin sectioning by ultramicrotomy can successfully be combined with subsequent confocal Raman imaging. This new 3D Raman imaging technique reaches a depth resolution of up to 100 nm, which is about 10‐fold better than in confocal Raman microscopy. Structurally complex and optically inhomogeneous samples can not only be imaged, but also be used to quantify structural parameters. Serial section Raman tomography is a promising method for materials science and possibly also for life sciences. [ABSTRACT FROM AUTHOR]

Risk-reducing salpingo-oophorectomy (RRSO) is recommended for women at increased risk of ovarian, fallopian tube (FT), and peritoneal carcinoma (collectively OC). We describe rates of occult neoplasia in the largest single-institution prospective cohort of women undergoing RRSO, including those with mutations in non- BRCA homologous repair (HRR) genes. Participants undergoing RRSO enrolled in a prospective tissue bank between 1999 and 2017. Ovaries and FTs were serially sectioned in all cases. Participants had OC susceptibility gene mutations or a family history suggesting OC risk. Analyses were completed in Stata IC 15.1. Of 644 women, 194 (30.1%) had mutations in BRCA1 , 177 (27.5%) BRCA2 , 27 (4.2%) other HRR genes, and 15 (2.3%) Lynch Syndrome-associated genes. Seventeen (2.6%) had occult neoplasms at RRSO, 15/17 (88.2%) in the FT. Of BRCA1 carriers, 14/194 (7.2%) had occult neoplasia, 8/194 (4.1%) invasive. One PALB2 and two BRCA2 carriers had intraepithelial FT neoplasms. Occult neoplasm occurred more frequently in BRCA1 / 2 carriers ≥45 years of age (6.5% vs 2.2%, chi square, p =.04), and 211/371 (56.9%) BRCA1/2 carriers had surgery after guideline-recommended ages. Four in 8 (50%) invasive and 2/9 (22%) intraepithelial neoplasms had positive pelvic washings. None with intraepithelial neoplasms developed recurrence or peritoneal carcinoma. BRCA1 carriers have the highest risk of occult neoplasia at RRSO, and the frequency increased with age. Women with BRCA1/2 mutations often have RRSO beyond recommended ages. One PALB2 carrier had FT intraepithelial neoplasia, a novel finding. Serial sectioning is critical to identifying occult neoplasia and should be performed for all risk-reducing surgeries. • BRCA1 mutation carriers had the highest frequency of occult neoplasia at time of risk-reducing salpingo-oophorectomy (7.2%). • The frequency of occult neoplasia was higher in older BRCA1 and BRCA2 mutation carriers. • Nearly 60% of BRCA1 and BRCA2 mutation carriers had risk-reducing surgery after guideline-recommended ages. • A PALB2 mutation carrier had an occult fallopian tube neoplasm, highlighting need for serial sectioning for all at risk. [ABSTRACT FROM AUTHOR]

2D sections of rice (Oryza sativa L.) mesophyll cells are complex and the cell interior contains a large volume of chloroplasts. Furthermore, the structure of chloroplasts changes in response to salt stress. In this study, we used a 3D reconstruction method based on serial section light microscopy to analyze a wide range of structures in leaf tissues and compared the intracellular structure of mesophyll cells of rice under normal and salt-treated conditions. The 3D reconstructed models revealed that mesophyll cells appeared as ellipsoid discs with several lobes around the cell periphery, with the volumes showing no significant difference between control and salt-treated plants. The chloroplast structure in the whole mesophyll cell was altered under salt stress, showing a reduced coverage area, whereas their volumes did not differ between treatments. These findings suggest that 3D reconstruction based on serial light micrographs can reveal the morphology of cells and chloroplasts in plant tissue. [ABSTRACT FROM AUTHOR]

This article is a Commentary on Harwood et al. (2020), 225: 2567–2578. [ABSTRACT FROM AUTHOR]

Development of the Digital Material Representation (DMR) model, based on 3D reconstruction algorithm and serial sectioning, is the main goal of the present paper. Details on the serial sectioning and image processing algorithms are presented first. Serial sectioning is realized on the basis of light microscopy (LM). Then concept of 3D reconstruction and developed algorithms are presented. Two approaches, based on shape coefficients and the flood fill algorithms, are developed to identify corresponding features on subsequent 2D images. Then, the interpolation algorithm to reconstruct 3D volume between 2D images is presented. Finally, obtained 3D model is an input for finite element mesh generation software for subsequent finite element calculations.

This work represents an interesting development in the detection and interpretation of crack evolution in through hole technology (THT) solder joints, which based on the development of general and common method. Serial sectioning is a useful method because it overcomes the problems associated with traditional two-dimensional metallographic techniques by providing information about (micro)structures in three-dimensions.

Serial sectioning electron microscopy (EM) of millimeter-scale three-dimensional (3D) anatomical volumes requires the collection of thousands of ultrathin sections. Here, we report a high-throughput automated approach, GAUSS-EM (guided accumulation of ultrathin serial sections-EM), utilizing a static magnetic field to collect and densely pack thousands of sections onto individual silicon wafers. The method is capable of sectioning hundreds of microns of tissue per day at section thicknesses down to 35 nm. Relative to other automated volume EM approaches, GAUSS-EM democratizes the ability to collect large 3D EM volumes because it is simple and inexpensive to implement. We present two exemplar EM volumes of a zebrafish eye and mouse olfactory bulb collected with the method., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Highlights • 3D imaging at high spatial resolution with electron microscopes. • Summary of the current state of the art of volume microscopy. • Serial sectioning, serial-block face scanning microscopy and focussed ion beam scanning microscopy. • Focus on sample preparation for volume microscopy. Abstract Volume microscopy has become an important method in cellular biology. In contrast to tedious serial sectioning volumes can now far more conveniently be obtained with serial-block face and focussed ion beam scanning electron microscopy. Serial-block face scanning electron microscopy is the instrument of choice for large volumes whereas focussed ion beam scanning electron microscopy has its merits in high voxel resolution. These aspects are discussed along with some specific applications of a focussed ion beam scanning electron microscope. [ABSTRACT FROM AUTHOR]

Mechanosensory organelles (MOs) are specialized subcellular entities where force-sensitive channels and supporting structures (e.g., microtubule cytoskeleton) are organized in an orderly manner. The delicate structure of MOs needs to be resolved to understand the mechanisms by which they detect forces and how they are formed. Here, we describe a protocol that allows obtaining detailed information about the nanoscopic ultrastructure of fly MOs by using serial section electron tomography (SS-ET). To preserve fine structural details, the tissues are cryo-immobilized using a high-pressure freezer followed by freeze-substitution at low temperature and embedding in resin at room temperature. Then, sample sections are prepared and used to acquire the dual-axis tilt series images, which are further processed for tomographic reconstruction. Finally, tomograms of consecutive sections are combined into a single larger volume using microtubules as fiducial markers. Using this protocol, we managed to reconstruct the sensory organelles, which provide novel molecular insights as to how fly mechanosensory organelles work and are formed. Based on our experience, we think that, with minimal modifications, this protocol can be adapted to a wide range of applications using different cell and tissue samples. Key features • Resolving the high-resolution 3D ultrastructure of subcellular organelles using serial section electron tomography (SS-ET). • Compared with single-axis tilt series, dual-axis tilt series provides a much wider coverage of Fourier space, improving resolution and features in the reconstructed tomograms. • The use of high-pressure freezing and freeze-substitution maximally preserves the fine structural details., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY-NC license.)

As obligate intracellular parasites, viruses need to hijack their cellular hosts and reprogram their machineries in order to replicate their genomes and produce new virions. For the direct visualization of the different steps of a viral life cycle (attachment, entry, replication, assembly and egress) electron microscopy (EM) methods are extremely helpful. While conventional EM has given important information about virus-host cell interactions, the development of three-dimensional EM (3D-EM) approaches provides unprecedented insights into how viruses remodel the intracellular architecture of the host cell. During the last years several 3D-EM methods have been developed. Here we will provide a description of the main approaches and examples of innovative applications.

Abstract Dendrochronology mostly deals with secondary (radial) growth and attention to primary (height) growth has so far been limited. However, tree-ring widths might not adequately represent stem volume increments, net primary productivity and the size of the tree stem carbon sink. The main reason for the prevailing focus on radial growth is that establishing height growth chronologies requires time-consuming and destructive methods. However, for certain ecological applications, less laboriously acquired data on height growth averaged over several successive years are satisfactory. Here we present an R package that contains a set of tools for the analysis of height growth. The tools have been developed for input data of tree-ring widths extracted from series of successive stem height levels. Tree-ring widths ideally represent four directions in each cross section to capture potential changes in stem eccentricity between various height levels. The main computed parameters provided by the package include height growth along the stem, changes of stem eccentricity and stem taper. Accurate determination of average height growth depends on the correct estimation of the number of tree rings at different stem height levels, which might be complicated by missing rings in off-pith cores. The presented package therefore also contains functions implementing common procedures for the estimation of the number of missing tree rings near to the pith. Most outputs can be visualized graphically. The package is useful for estimating height growth in ecological and dendrogeomorphological studies, especially in situations where both primary and secondary growth is influenced by different environmental factors. It is also useful for analysing tree-ring chronologies assembled using serial sectioning, which typically applies to shrubs. [ABSTRACT FROM AUTHOR]

The quantitative microstructural analysis in recrystallized pure iron is carried out by serial sectioning experiment coupled with three-dimensional (3-D) grain reconstruction technique. The full 3-D morphology of 16254 pure iron grains, which is the largest experimental 3-D grain dataset to date, is obtained. It is shown that the peak and the mean of the grain face number distribution were 10 and 13.5, respectively. For individual grains, the average trends that the highest and lowest face classes exhibited the highest affinity for mutual contact but the lowest affinity for contact with themselves are experimentally confirmed by 3-D pure iron grains. The pure iron grains are observed to have much more three-edged faces than Monte Carlo grains, resulting in relatively higher average dispersions of the grain edge distribution. [ABSTRACT FROM AUTHOR]

Abstract Three-dimensional (3-D) Monte Carlo simulations of grain growth with sub-boundaries in the presence of precipitates were performed based on the distribution of realistic grain boundary energy data. The simulated grains with sub-boundaries grew abnormally. The simulated and experimental morphologies of the abnormally growing grains in Fe-3%Si steel were compared. The experimental 3-D morphology was reconstructed from serially sectioned images. Both the simulated and experimental 3-D morphologies of abnormally growing grains have highly irregular and complicated shapes whereas both the simulated and experimental 3-D morphologies of normally growing grains have relatively regular and simple shapes. Growth by wetting is responsible for the highly irregular shape of the experimental abnormally growing grains of Fe-3%Si steel as well as for the highly irregular shape of the simulated abnormally growing grains. Highlights • Grains with sub-boundaries grew abnormally in the MC simulation with realistic grain boundary energy. • The experimental 3-D morphology of Goss grain was reconstructed from serially sectioned images. • 2-D sectioned microstructures of the MC simulation and experiment have highly irregular shape. • Both simulated and experimental 3-D morphologies of AGG have similar complicated shapes. [ABSTRACT FROM AUTHOR]

In this study, a range of advanced techniques have been used to characterise iodine-induced stress corrosion cracks, and investigate their interaction with microstructure in zirconium alloys used as cladding material in nuclear power reactors. C-ring samples, machined from cladding tubes in a recrystallised and cold worked condition, were subjected to compressive stress while submerged in an iodine-ethanol solution to simulate stress corrosion cracks arising from pellet cladding interaction. The complex morphology of these stress corrosion cracks was imaged in 3D using X-ray computed tomography (XCT), and subsequent analysis using both 2D and 3D electron backscatter diffraction (EBSD) allowed for the interaction of these cracks with microstructure to be investigated. Intergranular cracking modes were observed to dominate in the recrystallised microstructure, resulting in cracking in the radial direction with variations on the order of the grain size. Crack propagation in the cold worked microstructure was observed to be correlated with the direction of the elongated grains, resulting in a more tangential crack path with less local variation and a microstructure potentially more susceptible to iodine-induced stress corrosion cracking. 3D-EBSD using serial sectioning allows for the determination of the full five-parameter grain boundary description, and opens up the possibility of a statistical analysis of susceptible boundaries to SCC. Nanoscale secondary ion mass spectrometry (NanoSIMS) was also used to map the chemical distribution in the crack tip region and provides possible evidence of iodine segregation ahead of the crack tip. The results provide new insight into crack propagation mechanisms in recrystallised and cold worked microstructures, and pave the way for a more mechanistic understanding of crack propagation through advanced characterisation of iodine-induced stress corrosion cracks in zirconium alloys. [ABSTRACT FROM AUTHOR]