Microscopy and Microanalysis

Cryo-Focused Ion Beam Scanning Electron Microscopy (cryoFIB-SEM) using samples fixed by high-pressure freezing uniquely enables high resolution cryo-volume Electron Microscope (cvEM) images of cell ultrastructure to be obtained from whole cells and complex tissues in their near native state. As the freezing process also preserves fluorescence, the link between three-dimensional (3D) ultrastructure and biological process is also enabled by targeted cryo-Correlative Light and Electron Microscopy (CLEM). However, the overall viability of cvEM is challenged by sample preparation, charge balance during imaging, sample sensitivity to beam damage, contamination, and very long acquisition times. Here we detail new experimental workflows to significantly reduce each of these effects and demonstrate the improvement in resolution possible with results from the nematode Caenorhabditis elegans and the ciliated protozoon Paramecium bursaria containing many endosymbiotic algae. These results demonstrate the versatility and potential wide-ranging utility of cvEM for 3D ultrastructural imaging of whole multicellular and unicellular organisms.

Calculation of density maps from atomic models is essential for structural studies using crystallography and electron cryo-microscopy (cryoEM). These maps serve various purposes, including atomic model building, refinement, visualization, and validation. However, accurately comparing model-calculated maps to experimental data poses challenges, particularly because the resolution of cryoEM experimental maps varies across the map. Traditional crystallography methods generate finite-resolution maps with uniform resolution throughout the unit cell volume, while most modern software in cryoEM employ Gaussian-like functions to generate these maps, which does not adequately account for atomic model parameters and resolution. Recent work by Urzhumtsev & Lunin (2022, IUCr Journal, 9, 728-734) introduces a novel method for computing atomic model maps that incorporate local resolution and can be expressed as analytically differentiable functions of all atomic parameters. This approach enhances the accuracy of matching atomic models to experimental maps. In this paper, we detail the implementation of this method in CCTBX and Phenix. SynopsisNew tools implemented in CCTBX and Phenix allow the calculation of variable-resolution maps through a sum of atomic images expressed as analytic functions of all atomic parameters, along with their associated local resolution.

Bacteria microcompartments (BMCs) are pseudo-organelles comprised of a self-assembling, semi-permeable protein shell, most commonly enclosing components of enzymatic pathways. -Carboxysomes (-CBs) are anabolic BMCs known for their role in sequestering Rubisco, the enzyme responsible for carbon fixation in plants, algae and bacteria, along with an upstream enzyme and an assembly protein. Rubisco has low selectivity for its substrate, CO2, and has a slow enzymatic turnover rate, resulting in an inefficient metabolic pathway. Within the -CB, Rubisco has been observed at a range of concentrations and with either a liquid-like assembly or a pseudo-lattice of polymerized fibrils. The biophysical origins of the fibril ultrastructure organization are unclear; however, it is only observed inside -CBs. Quantitative knowledge of the binding constants and energies for assembly and maintenance of these fibrils is critical for understanding this organization and Rubisco regulation, but quantitative methods for in situ analysis of Rubisco polymerization have been lacking. Here, we present an approach to convert tomography-derived -CB volumes and Rubisco particle positions into polymerization binding curves. We used this procedure to determine the Rubisco polymerization constants, including the nucleus size (n) and equilibrium polymerization constant (Kpol). The adopted modeling approach is consistent with in situ constraints, such as concentration-dependent binding interactions and confinement. This approach offers a powerful tool to evaluate both in vitro and potentially in vivo biomolecular interactions, both of Rubisco and of other proteins and polymers suitable for analysis by cryo-electron tomography. Significance StatementCryogenic electron tomography (cryoET) is a powerful method to resolve structures of proteins in their native environment at subnanometer-level resolution. Because tomography data retains spatial relationships of all particles, it intrinsically contains information about component (e.g., protein) binding interactions. Here, we use Rubisco polymerization in -carboxysomes as a model system to demonstrate that quantitative, biochemical binding analysis is possible with cryoET.

Affinity chromatography is a powerful and therefore popular method for the purification of proteins for structural studies. The success of the technique relies on the specificity of the interaction between the target protein and the affinity resin. Here, we present the identification of two protein contaminants isolated from HEK293 cell lysate following affinity purification of twin Strep-tagged or FLAG-tagged proteins. The contaminants were identified as human propionyl-coenzyme A carboxylase (hPCC) and protein arginine methyltransferase 5 in complex with methylosome protein 50 (PRMT5:MEP50) via a combination of cryo-EM data processing and proteomic analyses. This report serves to illustrate how these contaminants may appear in cryo-EM datasets and to highlight the paramount importance of affinity chromatography resin specificity for efficient protein purification.

Dermal bone, which forms a variety of skeletal structures and persists in a wide range of extant vertebrates, evolved prior to endochondral bone which forms all mammalian load-bearing bones. Sturgeons are a family of fish which diverged soon after the lobe-finned/ray-finned split. Sturgeon retain a long robust spine at the leading edge of the pectoral fin, called the pectoral fin spine (PFS). Pectoral fin spines are bone elements that are present in many extinct and extant species of non-tetrapod jawed fish. In this study, we characterize the structure (light, polarized, micro-computed tomography and scanning electron microscopy), composition (FTIR, TGA, BMD), and mechanical properties (3-point bending and microindentation) of the pectoral fin spine (PFS) of the Russian sturgeon (Huso gueldenstaedtii). The microstructure of the PFS is highly organized as it is formed by dermal osteonal bone and parallel fibered bone. Its microarchitecture, along with high material toughness, anisotropy, and substantial ash content, enables the PFS to bear loads and function in both locomotion and protection. In addition, we show an interconnected network of neurovascular canals and ornamentations, features also found in pectoral fin spines of other non-tetrapod jawed fish. Collectively, these findings demonstrate that dermal bone can form structurally organized, mechanically competent load-bearing elements and provide new insight into pectoral fin spines in ray-finned fish.

Fourier ptychographic microscopy (FPM) uses sequential multi-angle illumination and iterative phase retrieval to recover a high-resolution complex image from a series of low-resolution brightfield and darkfield images. We present OpenFPM, an open-source FPM platform in which conventional and optomechanical hardware is replaced with compact, low-cost 3D printed components. Illumination, sample and objective positioning, and camera triggering are controlled using a Python-based interface on a Raspberry Pi microcomputer. With a 10 x /0.25 NA objective lens and 636 nm illumination, OpenFPM experimentally achieves amplitude and phase reconstructions with an effective synthetic NA of 0.90 over a 1 mm field-of-view. This platform gives researchers accessible and affordable hardware for developing and testing LED-array microscopy techniques for a range of biomedical imaging applications.

Recent advances in high-resolution imaging and spatial transcriptomics have enabled reconstruction of complex 3D tissue maps, providing unprecedented insights into cellular connectivity, organization, and tissue architecture. However, standardization challenges hinder integration, sharing, and analysis of these datasets across research communities. We developed NetTracer3D to simplify three-dimensional image analysis across diverse datasets. NetTracer3D is an integrated tool for defining, processing, and sharing 3D tissue maps with standardized data formats and interactive exploration capabilities. It provides three broadly applicable network analysis modalities: Connectivity networks for analyzing functional tissue units or cells connected via secondary structures such as nerves or vasculature; Branch Adjacency and Branchpoint networks for converting branched anatomical structures into analyzable representations; and Proximity networks for grouping structures by spatial relationships to identify cellular organization patterns. We demonstrate several use cases applying NetTracer3D to analyze multidimensional data from CODEX and label free Raman spectroscopy, multiscalar data encompassing subcellular and anatomical scales and a range of modalities. NetTracer3D was able to characterize neural relationships between functional tissue units in human and mouse kidneys and mouse bronchi. Branchpoint networks were used to identify vascular defects in human brain angiogram and define the innervation structure of a lymph node. Finally, we demonstrate how proximity networks characterize the tumor microenvironment in 3D light sheet cancer images and auto-detect cellular neighborhoods in multiplexed 2D CODEX datasets. Beyond network creation, NetTracer3D enables analysis, spatial statistics, and visual analytics tailored for volumetric tissue data. By establishing interoperable formats and analysis workflows, this work provides accessible and reproducible analytical tools for 3D spatial biology, enabling new discoveries of relationships between structure and physiology.

Coccolithophores are unicellular marine phytoplankton that produce complex and intricately shaped mineralised scales called coccoliths. Coccoliths are produced in an intracellular vesicle where crystal nucleation occurs, from which several individual calcite units develop with anisotropic crystallographic facets, prompting studies into the cellular mechanisms which control crystal growth within the cell. Here, we characterise those morphological developments in 3D that occur during the formation of coccoliths by the species Gephyrocapsa huxleyi using cryo-ptychographic X-ray computed tomography. This technique is ideally suited to study coccolith mineral development, as intracellular structures can be imaged intact in their native state without needing to disrupt cells. Combined with additional imaging of developing coccoliths using cryo-transmission electron microscopy and scanning electron microscopy, we report the developmental stages involved in coccolith growth across the complete mineralisation period, while also showing that the constrained space created by individual crystal units growing in close confinement affects the final crystal morphology and overall mineral structure. These findings provide clarification on the mineralisation pathways that coccolithophores and other biomineralising organisms use to control the formation of highly functionalised crystalline structures, particularly relevant in the design of materials with tunable properties.

Dragonfly and cicada wing-inspired titanium nanopillar surfaces show promising bactericidal properties for antibacterial medical implant applications, but the precise mechanisms of bacteria-nanopillar interactions under hydrated conditions remain unclear. Cryo-electron tomography (cryo-ET) enables the visualisation of cellular organelles within their native hydrated cellular environment at molecular resolution. Visualising the bacteria-material interface on nanostructured surfaces by cryo transmission electron microscopy (cryo-TEM) requires the preparation of thin lamellae. Obtaining lamellae of bacteria directly on metal substrates while in a non-fixed and hydrated state requires cryo-focused ion beam (cryo-FIB) milling to isolate the targeted bacteria from the bulk sample. This approach faces additional challenges compared to tissues or cells on TEM grids, as titanium samples require a simultaneous cross-section of soft and hard materials at the same position and require vitrification, which embeds the sample in a thick layer of ice. Nonetheless, we demonstrate how to target a specific bacterium interacting with a titanium nanopillar surface using correlative cryo-fluorescence imaging, and how lamellae can still be prepared from vitrified samples by extracting the targeted bacterium and its surrounding as a small volume and transferring it to a receptor grid for thin lamella preparation, called targeted cryo-lift-out. Here, we outline the workflows and discuss their advantages and limitations for producing lamellae through lift-out techniques under cryogenic conditions, using methods that do not involve gas injection systems (GIS) for the lift-out transfer. These advances enhance cryo-ET applications, enabling in situ investigations of the interface between bacteria and nanopillars to effectively study the bactericidal mechanisms of biomimetic nature-inspired nanotopographies in a hydrated environment.

In wildlife forensic practice, species identification and estimation of the Minimum Number of Individuals (MNI) for highly processed specimens have long relied on weight-based conversion methods, which may result in underestimation of the number of individuals involved in a case. Focusing on confiscated casque products of the helmeted hornbill (Rhinoplax vigil), this study combines macroscopic morphological examination with mitochondrial DNA barcoding (16S rRNA, COI, and Cytb) to explore a more robust approach for individual quantification. The results demonstrate that the conventional "weight-based" approach overlooks critical biological information contained in anatomical structures and cannot accurately reflect the actual number of individuals involved. Based on this, we propose an anatomy-based criterion centered on the principle of structural uniqueness: specimens retaining biologically unique beak or casque structures should be directly assigned to a single individual, whereas weight-based estimation should only be applied when original anatomical features are entirely absent. In addition, considering material loss during processing, we propose approximately 85 g as a reference threshold for estimating the number of individuals in heavily processed solid casque products. This approach improves the scientific rigor and accuracy of forensic identification and provides reliable technical support for the conviction, sentencing, and law enforcement of wildlife trafficking cases involving helmeted hornbill and other endangered species.

Single-molecule localization microscopy (SMLM) methods enable fluorescence imaging of biological specimens with nanometer-scale resolution. Although fluorophore localization precision is theoretically limited only by photon statistics, in practice the resolution of SMLM images is often degraded by physical drift of the sample and/or the microscope during data acquisition. At present, correcting this effect requires either specialized stabilization systems or computationally intensive post-processing, and established drift correction algorithms based on image cross-correlation suffer from limited temporal resolution. In this study we introduce COMET, a new method for SMLM drift estimation which achieves a substantially higher precision, accuracy, and temporal resolution compared with existing algorithmic approaches. We demonstrate that improved drift estimation translates directly into higher SMLM image resolution, limited by localization precision rather than drift artifacts. COMET is applicable to all types of SMLM data, operating directly on 2D or 3D localization datasets, and is readily integrated into analysis workflows. We benchmark its performance using both simulations and experiments, including STORM, MINFLUX, and Sequential OligoSTORM measurements, where long acquisition times make drift correction particularly challenging. COMET is published as an open-source, Python-based software project and is also available on open cloud-computing platforms.

Cortical organoids (COs) represent a powerful in vitro model system that recapitulates key aspects of human brain development, enabling the study of neurodevelopmental processes, cellular diversity, and disease mechanisms in a physiologically relevant 3D environment. However, traditional histological analysis of COs relies on tissue sectioning, which limits the ability to capture the full spatial complexity of organoid architecture. In this study, we establish a framework for applying CLARI-O, an improved tissue-clearing technique, for intact COs and organoid-based systems, enabling comprehensive 3D visualization and analysis of 3D organizational features. Using CLARI-O in combination with high-resolution imaging, we demonstrate the utility of tissue clearing for studying glial populations, including oligodendrocytes and microglia, considered to be underrepresented in COs, and their interactions with neurons. Additionally, we apply this method to forebrain assembloids (FAs) to visualize cellular heterogeneity and the interface between ventral and dorsal regions. Finally, we use CLARI-O to study mouse brains containing xenotransplanted COs (MB-COs) to evaluate human cell integration, migration, vascularization, and structural connectivity. This is the first study to demonstrate how tissue clearing can be used after functional assays such as calcium imaging to correlate neural activity with post hoc structural analysis in MB-COs. Together, this work establishes CLARI-O as a powerful tool for advancing 3D structural and functional interrogation of human CO-derived systems, enhancing their value for disease modeling, drug screening, and translational neuroscience. MotivationCortical organoids have become an increasingly powerful tool in neuroscience. Their complexity has expanded substantially, now incorporating exogenous lineages, fusing organoids with distinct regional identities (assembloids), and enabling xenotransplantation into in-vivo environments. These advancements require more sophisticated technological approaches that are capable of capturing the intricate three-dimensional cyotarchitecture and organization of intact organoid systems both in vitro and after xenotransplantation in vivo. Tissue-clearing methodologies offer a unique opportunity to visualize these structural and cellular features with exceptional depth and resolution. Graphical abstract HighlightsO_LIWe optimized clearing protocols to develop an organoid specific clearing method (CLARI-O) that enables high-resolution visualization of diverse neuronal and glial populations without tissue sectioning, preserving long-range connections and cellular processes. C_LIO_LIForebrain assembloids used to study neuronal and oligodendrocyte migration can be effectively processed using CLARI-O, allowing detailed visualization of fusion interface. C_LIO_LIWe established a robust framework for CLARI-O-based clearing of mouse brain tissue containing xenotransplanted human cortical organoids, enabling comprehensive 3D analysis of graft development, integration, and vascularization in vivo. C_LI

Digital holographic microscopy systems in a common-path configuration, compared to systems with a separate reference arm, offer a compact design and resistance to disturbances. They can operate with partially coherent illumination, reducing speckle noise. However, they are limited by the overlapping of the object beam and its laterally shifted replica. As a result, images from different regions of the object overlap on the detector, preventing imaging of dense samples. We present the wavelength-scanning replica-removal method, which solves this problem by enabling the separation of information from both replicas and thereby doubling the effective field of view (FOV). The wavelength-scanning multi-shear replica removal algorithm plays a key role in reconstructing the undisturbed phase from a series of holograms recorded with variable shears. The shear value is controlled by changing the illumination wavelength. This enabled the development of two measurement modes: time-domain wavelength scanning for high-quality imaging, and a single-shot mode with frame division into color channels to improve temporal resolution. The method was validated using resolution tests and biological samples - neurons and dynamic yeast cultures. By combining the advantages of the common-path configuration with dense-structure imaging and dynamic processes, the proposed method constitutes a versatile tool for quantitative phase microscopy.

Quantifying chromatin-state dynamics in living cells remains challenging, in part because most methods require fixation or cell lysis. Here, we benchmark and introduce three simple live-cell image-derived metrics computed from routine DNA staining--the coefficient of variation (CV), 1-Gini, and the Diffuse Signal Index (DSI), introduced here--as fixation-free readouts of chromatin state. Using HL60-derived neutrophils (dHL-60) undergoing NETosis as a model system with a pronounced compact-to-decompact chromatin transition, we show that all three metrics track progressive chromatin reorganization in live-cell trajectories, but differ markedly in sensitivity: DSI provides the strongest trajectory-level discrimination between NETing and non-NETing cells, followed by 1-Gini and CV. Comparison with Tn5-based chromatin accessibility measurements in fixed cells further shows that all three metrics correlate with chromatin accessibility, supporting their biological relevance. Together, our results provide a practical framework for extracting chromatin-state readouts from routine live-cell DNA staining and identify DSI as the most discriminative metric for tracking chromatin reorganization in this benchmark.

Ligand binding has been shown to induce significant alterations in the conformational landscape of proteins. Traditional crystallography approaches have provided valuable input about the end states in ligand-binding reactions. However, dynamical relationships between ligand binding and backbone rearrangement often remain obscured by crystallographic structures. In the present study, we use time-resolved serial synchrotron crystallography (TR-SSX) to directly visualize indole binding in the cavity of T4 lysozyme L99A in microcrystals under controlled environmental conditions. By integrating fixed target crystallography with LAMA-based ligand delivery, we have been able to track the progression of ligand binding and backbone rearrangement. By utilizing an occupancy refinement protocol, we have been able to quantify structural populations. Our studies reveal that ligand binding for this protein cavity follows a diffusion-limited process that progressively rearranges the F -helix of the protein towards a dominant conformational state. These findings establish an observable link between ligand diffusion, occupancy evolution and conformational adaptation within a crystalline environment. More broadly, our work shows how TR-SSX can quantify ligand and conformational populations during binding, providing a framework to interpret structural adaptation in real time.

Magnetoreception, the ability to perceive the geomagnetic field, is widespread across animals. The underlying sensory mechanism remains elusive, but a long-standing hypothesis proposes single-domain magnetite linked to mechanosensitive ion channels. The Ansells mole-rat (Fukomys anselli) is a subterranean rodent with a magnetic sense, and published behavioral and histological data are consistent with magnetite-based magnetoreceptors in the cornea or retina. Here, we systematically screened for magnetite in the mole-rat eye, combining iron detection via enhanced Prussian blue staining and synchrotron X-ray fluorescence microscopy (XFM) with magnetic detection via MRI quantitative susceptibility mapping (MRI-QSM) and quantum-diamond microscopy (QDM). This revealed only a few iron particles in the retina and cornea, which predominantly overlapped with titanium or chromium, indicating a non-biogenic origin. XFM showed iron-enriched lines in the cornea, but these did not show ferrimagnetic signals. Focusing on other ocular tissues, MRI-QSM revealed the highest susceptibility in the ciliary body, where iron-rich pigmented cells were identified. A TEM-screen, however, failed to detect single-domain magnetite particles in these cells. We conclude that our high-sensitivity multimodal screen provides no evidence for magnetite-based magnetoreceptors in the mole-rat eye, suggesting that mole-rat magnetoreceptors either do not reside in the eye or are based on different physical principles.

Domestic dogs (Canis familiaris) display more morphological variation than any other mammal. Cranial morphology has been extensively studied, as have the relationships with function, development, genetics, veterinary medicine, and breed welfare. Postcrania remain comparatively understudied, despite well-documented breed-specific predispositions to musculoskeletal disease. Here, we apply three-dimensional landmark-free morphometrics to quantify the shape of 743 elements from 213 dogs, including the scapula, humerus, radius, ulna, pelvic girdle, femur, tibia, and fibula. We assess integration among limb elements and investigate drivers of shape variation within and between breeds. Across most breeds, limb bone shape is strikingly similar. Dachshunds, however, exhibit distinct morphology across all elements and one to two orders of magnitude greater variation than any other breed. Despite this disparity, integration remains high between all element pairs. Remarkably, we find no significant relationship between bone shape and body mass, age, or pathology, but comparison with historic specimens reveals marked changes in dachshund long bone shape over the past [~]150 years. These extreme differences are not shared by other sampled chondrodysplastic breeds, underscoring the need to understand morphological diversity beyond simple categorisation. These findings provide a quantitative framework for linking postcranial morphology with function, disease risk, and evidence-based improvements to canine welfare.

Capturing meta-stable conformations of enzymes and ligand complexes demands structural snapshots beyond static crystal structures. While time-resolved serial crystallography at room temperature, offers a time-resolution down to the femto-second domain it requires large amounts of micro crystals, specialized beamlines and considerable experience. Moreover, as the majority of enzymes displays turnover-times in the millisecond domain or slower, simpler methods can provide meaningful structural insight into enzyme catalysis. Vitrification of protein crystals can trap reaction intermediates by rapid cooling to {inverted exclamation} 100 K, and has traditionally been used to gain insight into long lived reaction intermediates such as product complexes. However, manual vitrification procedures are limited to long delay times of at least several seconds and heavily suffer from operator variability. A solution to this problem is provided by automatic crystal plunging devices, such as the Spitrobot, that plunge loop-mounted protein crystals into liquid nitrogen within millisecond time-scales. Versatile means of reaction initiation can be achieved either by micro dispensing a ligand droplet, or via optical excitation of light-sensitive proteins, or via the photoactivation of caged compounds. In addition to the conceptual simplicity, another benefit of cryo-trapping is that data can be collected at conventional synchrotron beamlines, exploiting their robust high-throughput capabilities. Thus, compared to room-temperature time-resolved crystallography, users not only benefit from uncoupling sample-preparation and data-collection, but also from a reduction in the required technical expertise and ready access to radiation sources. However, as cryo-trapping crystallography explores dynamic structural changes that become only visible by the comparison of several samples, experiments have to be carefully planned to carry out the necessary controls and to avoid mis- or over-interpretation of the results. Here we describe a detailed protocol for cryo-trapping time-resolved crystallography using automated crystal-plungers that enables researchers to map enzymatic reaction coordinate pathways within the millisecond domain.

The spatiotemporal organisation of chromatin in the eukaryotic nucleus is fundamental for genome regulation. Chromatin undergoes rapid remodelling and rearrangements within minutes, altering its diffusion properties. Considering the tight coupling between genome function and nuclear architecture, a key question is how chromatin dynamics adapt to or promote nuclear processes. To elucidate the underlying physical principles, we employed High-resolution Diffusion mapping (Hi-D) to track chromatin motion throughout interphase in live human cells. Our analysis, that considers both diffusive motion and drift generated by active forces, revealed that chromatin dynamics are heterogeneous, with distinct behaviours in different subnuclear zones. Notably, both diffusive and processive contributions to chromatin motion progressively decrease from G1 to G2 phase, with this reduction occurring uniformly across all subzones. This suggests a global mechanism driving the observed decrease in chromatin mobility during cell cycle progression. By combining genetic knockout experiments and polymer modelling, we demonstrate that the doubling of DNA content, rather than cohesin-mediated sister chromatid entrapment, is responsible for the gradual decrease in chromatin motion during the cell cycle in human nuclei. These findings provide new insights into the physical and functional organisation of chromatin and its regulation during cellular proliferation. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/712877v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@75c654org.highwire.dtl.DTLVardef@2fbd3dorg.highwire.dtl.DTLVardef@31025aorg.highwire.dtl.DTLVardef@191808e_HPS_FORMAT_FIGEXP M_FIG C_FIG

We present a live-to-cryo correlative imaging workflow for multiscale structural and chemical analysis of biological tissues in their near-native state. The method integrates live super-resolution fluorescence microscopy, live and cryogenic Raman spectroscopy, and targeted cryogenic focused ion beam/scanning electron microscopy, transmission electron microscopy, electron tomography, energy dispersive X-ray spectroscopy, and electron diffraction. This approach enables precise 3D targeting and nanoscale imaging of selected regions across four orders of magnitude in spatial resolution, while preserving ultrastructure and chemical composition. Using regenerating zebrafish scales as a benchmark, we visualize collagen fibril orientation, local matrix density, and mineral composition within the extracellular matrix. We identify a plywood-like architecture of unmineralized collagen with orientation-independent density variation, and reveal curved, acidic phosphate-rich mineral platelets aligned with collagen fibrils. This workflow establishes a generalizable strategy for comprehensive 3D correlative analysis of hybrid tissues, and opens new opportunities for studying native structure-function relationships at the interface of biology and materials science.