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Achieving detailed neuronal structural information in large‐volume brain tissue has been a longstanding challenge in human brain imaging. A key obstacle arises from the trade‐off between staining efficiency and tissue autolysis. Traditional Golgi staining, typically conducted at room temperature or 37 °C to optimize staining efficiency, leads to rapid autolysis of brain tissue, resulting in the loss of fine structural details. Here, a near‐freezing temperature (NFT) staining strategy in post‐mortem frozen (PMF) human brain samples are presented, using a mercury chloride‐based method under ice‐water bath conditions. In contrast to the 37 °C Golgi staining, this NFT‐based method significantly reduces tissue autolysis, preserving fine neuronal structures. Notably, neuronal counts in the same field of view increased by 5.5‐fold, and dendritic spine density increases by 22‐fold. Using this approach, uniform staining of millimeter‐thick is achieved, centimeter‐scale human brain slices and integrated it with synchrotron‐based X‐ray microscopy to perform micrometer resolution 3D reconstructions of the cerebellum and frontal lobe. This novel technique offers a powerful tool for the fine‐structural imaging of large‐volume brain tissue, providing new insights into the intricate organization of neural networks. A near‐freezing temperature staining strategy, combined with a mercury chloride‐based method, effectively preserves delicate neuronal structures in post‐mortem frozen human brain tissues by minimizing autolysis. This approach enables uniform labeling across centimeter‐scale slices and, when integrated with synchrotron‐based X‐ray microscopy, achieves micrometer‐resolution 3D reconstructions, paving the way for large‐scale human brain mapping.

PREMISE OF THE STUDY: Recent phylogenetic analyses based on molecular data suggested that the monocot family Zingiberaceae be separated into four subfamilies and four tribes. Robust morphological characters to support these clades are lacking. Seeds were analyzed in a phylogenetic context to test independently the circumscription of clades and to better understand evolution of seed characters within Zingiberaceae. METHODS: Seventy-five species from three of the four subfamilies were analyzed using synchrotron based x-ray tomographic microscopy (SRXTM) and scored for 39 morphoanatomical characters. KEY RESULTS: Zingiberaceae seeds are some of the most structurally complex seeds in angiosperms. No single seed character was found to distinguish each subfamily, but combinations of characters were found to differentiate between the subfamilies. Recognition of the tribes based on seeds was possible for Globbeae, but not for Alpinieae, Riedelieae, or Zingibereae, due to considerable variation. CONCLUSIONS: SRXTM is an excellent, nondestructive tool to capture morphoanatomical variation of seeds and allows for the study of taxa with limited material available. Alpinioideae, Siphonochiloideae, Tamijioideae, and Zingiberoideae are well supported based on both molecular and morphological data, including multiple seed characters. Globbeae are well supported as a distinctive tribe within the Zingiberoideae, but no other tribe could be differentiated using seeds due to considerable homoplasy when compared with currently accepted relationships based on molecular data. Novel seed characters suggest tribal affinities for two currently unplaced Zingiberaceae taxa: Siliquamomum may be related to Riedelieae and Monolophus to Zingibereae, but further work is needed before formal revision of the family.

Understanding the structural and functional organisation of brain networks is a fundamental objective in neuroscience, with three‐dimensional (3D) reconstruction of single‐neuron morphology serving as a critical foundation. The Golgi staining method, which enables random neuronal labeling and provides high‐contrast signals in both optical and X‐ray microscopy, remains a valuable tool for morphological analysis. However, its widespread application in large‐scale neuronal reconstructions is hindered by signal discontinuities in neuronal branches, high‐density labeling, and complex background interference. While automated reconstruction methods perform well in sparsely labelled and morphologically simple neuronal populations, their effectiveness is limited in Golgi‐stained samples. Here we develop a semi‐automated single‐neuron reconstruction method for Golgi‐stained mouse brain neurons (SNR‐Golgi). By integrating three key technical modules—background denoising, single‐neuron extraction, and branch repair—SNR‐Golgi significantly enhances the accuracy and completeness of neuronal reconstruction. In fluorescence micro‐optical sectioning tomography (fMOST) datasets, SNR‐Golgi demonstrated superior performance in neuronal reconstruction within the mouse somatosensory cortex, achieving a 30% increase in reconstructed branch count, a 76% improvement in total branch length, and a 3.7‐fold increase in axonal length. Additionally, in synchrotron‐based X‐ray imaging datasets, SNR‐Golgi enabled submicron‐resolution 3D reconstruction of single neurons. These results demonstrate that SNR‐Golgi effectively addresses the complexity of Golgi‐stained samples and provides robust technical support for the structural analysis of brain neurons across various imaging modalities.

Abstract Background and Aims Understanding the spatial distribution of inorganic nutrients within edible parts of plant products helps biofortification efforts to identify and focus on specific uptake pathways and storage mechanisms. Methods Kernels of sweetcorn (Zea mays) variety ‘High zeaxanthin 103146’ and maize inbred line ‘Thai Floury 2’ were harvested at two different maturity stages, and the distributions of K, P, S, Ca, Zn, Fe and Mn were examined in situ using synchrotron-based X-ray fluorescence microscopy. Key Results The distribution of inorganic nutrients was largely similar between maize and sweetcorn, but differed markedly depending upon the maturity stage after further embryonic development. The micronutrients Zn, Fe and Mn accumulated primarily in the scutellum of the embryo during early kernel development, while trace amounts of these were found in the aleurone layer at the mature stage. Although P accumulated in the scutellum, there was no direct relationship between the concentrations of P and those of the micronutrients, compared with the linear trend between Zn and Fe concentrations. Conclusions This study highlights the important role of the embryo as a micronutrient reserve for sweetcorn and maize kernels, and the need to understand how biofortification efforts can further increase the inorganic nutrient concentration of the embryo for human consumption.

The quantification of elemental concentration in cells is usually performed by analytical assays on large populations missing peculiar but important rare cells. The present article aims at comparing the elemental quantification in single cells and cell population in three different cell types using a new approach for single cells elemental analysis performed at sub-micrometer scale combining X-ray fluorescence microscopy and atomic force microscopy. The attention is focused on the light element Mg, exploiting the opportunity to compare the single cell quantification to the cell population analysis carried out by a highly Mg-selective fluorescent chemosensor. The results show that the single cell analysis reveals the same Mg differences found in large population of the different cell strains studied. However, in one of the cell strains, single cell analysis reveals two cells with an exceptionally high intracellular Mg content compared with the other cells of the same strain. The single cell analysis allows mapping Mg and other light elements in whole cells at sub-micrometer scale. A detailed intensity correlation analysis on the two cells with the highest Mg content reveals that Mg subcellular localization correlates with oxygen in a different fashion with respect the other sister cells of the same strain.

Mechanical ventilation is not only a life saving treatment but can also cause negative side effects. One of the main complications is inflammation caused by overstretching of the alveolar tissue. Previously, studies investigated either global strains or looked into which states lead to inflammatory reactions in cell cultures. However, the connection between the global deformation, of a tissue strip or the whole organ, and the strains reaching the single cells lining the alveolar walls is unknown and respective studies are still missing. The main reason for this is most likely the complex, sponge-like alveolar geometry, whose three-dimensional details have been unknown until recently. Utilizing synchrotron-based X-ray tomographic microscopy, we were able to generate real and detailed three-dimensional alveolar geometries on which we have performed finite-element simulations. This allowed us to determine, for the first time, a three-dimensional strain state within the alveolar wall. Briefly, precision-cut lung slices, prepared from isolated rat lungs, were scanned and segmented to provide a three-dimensional geometry. This was then discretized using newly developed tetrahedral elements. The main conclusions of this study are that the local strain in the alveolar wall can reach a multiple of the value of the global strain, for our simulations up to four times as high and that thin structures obviously cause hotspots that are especially at risk of overstretching.

Methodological constraints limit the extent to which existing soil aggregation models explain carbon (C) stabilization in soil. We hypothesize that the physical infrastructure of microaggregates plays a major role in determining the chemistry of the occluded C and intimate associations between particulate C, chemically stabilized C and the soil mineral matrix. We employed synchrotron-based scanning transmission X-ray microscopy (STXM) coupled with near-edge X-ray absorption fine structure (C 1s-NEXAFS) spectroscopy to investigate the nanoscale physical assemblage and C chemistry of 150-micrometer microaggregates from a Kenyan Oxisol. Ultra-thin sections were obtained after embedding microaggregates in a sulfur block and sectioning on a cryo-microtome at -55°C. Principal component and cluster analyses revealed four spatially distinct features: pore surfaces, mineral matter, organic matter, and their mixtures. The occurrence of these features did not vary between exterior and interior locations; however, the degree of oxidation decreased while the complexity and occurrence of aliphatic C forms increased from exterior to interior regions of the microaggregate. At both locations, compositional mapping rendered a nanoscale distribution of oxidized C clogging pores and coating pore cavities on mineral surface. Hydrophobic organic matter of aromatic and aliphatic nature, representing particulate C forms appeared physically occluded in 2- to 5-micrometer pore spaces. Our findings demonstrate that organic matter in microaggregates may be found as either oxidized C associated with mineral surfaces or aromatic and aliphatic C in particulate form. Using STXM and C 1s-NEXAFS we are for the first time able to resolve the nanoscale biogeocomplexity of unaltered soil microaggregates.

Flowers of extant Quintinia are described in detail on the basis of field observations and serial microtome sections and compared with flowers of the Late Cretaceous Silvianthemum suecicum and Bertilanthus scanicus on the basis of new analyses of the fossil material. The analyses of both extant and fossil material also include synchrotron-based X-ray tomographic microscopy. The study shows that Quintinia and Silvianthemum/Bertilanthus have more features in common than previously recognized and strongly reinforces their earlier assumed close relationships. Among important characters linking the three genera are similar secretory hairs on the floral surface and pedicel; short sepals; free, quincuncial petals; broad anthers with small pollen sacs; 4-aperturate pollen (in Silvianthemum and Quintinia); postgenital union of the 3-4 apocarpous styles (in Silvianthemum and Quintinia); connection of the apocarpous carpel parts with the floral base; downward tapering ovary and locule(s); placentation on incomplete septa; and flask-shaped epidermal cells with a thin and long papilla on the carpels. Features observed in Quintinia, particularly the presence of bitegmic ovules and free petals, suggest that the apparent phylogenetic position with extant Paracryphiales (campanulids) needs further analysis. Bitegmic ovules are not known from other Paracryphiales or campanulids, and the presence of free petals is unusual in campanulids.

Synchrotron-based X-ray fluorescence microscopy (XFM) can localise chemical elements at a subcellular level. 99mTechnetium stannous (TcSn) colloid is taken up by phagocytes via a Complement Receptor 3 mediated phagocytic process. In the current study, XFM was used to examine the intracellular trafficking of TcSn colloid in neutrophils. XFM was performed on TcSn colloid, and neutrophils labelled with TcSn colloid, in whole blood. We developed a set of pixel by pixel analysis and mapping techniques incorporating cluster analysis that allowed us to differentiate neutrophils and artefactual contaminants, and we examined the changes in element distribution that accompany neutrophil phagocytosis of TcSn colloid. Sn became associated with half the neutrophils. Within cells, Sn colocalised with iron (Fe) and sulphur (S), and was negatively associated with calcium (Ca). Despite the high sensitivity of XFM, Tc was not detected. XFM can help clarify the intracellular processes that accompany neutrophil phagocytosis. The subcellular colocalisation of Sn with Fe is consistent with fusion of the colloid-containing phagosome with neutrophil granules. The association of Sn with S suggests that proteins rich in S-containing amino acids are present in the phagosome. The negative colocalisation with Ca indicates that ongoing maturation of the TcSn colloid phagosome is no longer calcium dependent one hour after phagocytosis.

A simple observation of the stress-strain curve (see Figure 7A in reference 5) of a silicone foam shows that stress relaxation of the polymer foam occurs as a result of the applied uniaxial compression. [...]in between compression steps, a pause of typically 5 to 15 minutes is needed before the tomogram is acquired. [...]generation synchrotron sources [10], such as Argonne National Laboratory’s Advanced Photon Source, can offer X-ray photon fluxes ~1012 to 1014 photons s-1, which is 3 to 5 orders of magnitude brighter than what is available in the laboratory. [...]this was not possible due to the location and length of the load stage’s power supply cord, which was located at the bottom base of the load stage. [...]an oscillating “washing machine” rotation was used. Large volumes of data were acquired for each sample imaged; reconstruction of a single tomogram resulted in a 4.8 GB file size, with a total of 96 GB per sample compression. [...]data reconstruction and data processing requires significant amounts of PC processing time and significantly depends on the hardware specifications of the processing PC.