Residual Stresses in Surgical Growing Rods.

The treatment of early onset scoliosis using surgical growing rods suffers from high failure rate. Fatigue resistance can be improved by inducing compressive residual stresses within the near surface region. An in-depth investigation of the residual stresses profile evolution is performed through the sequence of material processing steps followed by surgeons handling operations, in connection to material properties. The final goal is to guide further improvements of growing rod lifetime. Residual stress evaluation was carried out on Ti-6Al-4V rods using digital image correlation applied to microbeam ring-core milling by focused ion beam. This provided experimental stress profiles in shot-peened rods before and after bending and demonstrated that compressive residual stresses are maintained at both concave and convex rod sides. A finite element model using different core and skin conditions was validated by comparison to experiments. The combination of an initial shot peening profile associated with a significant level of backstress was found to primarily control the generation of compressive stresses at the rod surface after bending. Guidelines to promote larger compressive stresses at the surface were formulated based on a parametric analysis. The analysis revealed the first order impact of the initial yield strength, kinematic hardening parameters and intensity of the shot peening operation, while the bending angle and the depth of shot peening stresses were found to be of minor importance. Materials exhibiting large kinematic hardening and low yield strength should be selected in order to induce compressive residual stresses at key fatigue initiation site.

Hydrogel-Inducing Graphene-Oxide-Derived Core-Shell Fiber Composite for Antibacterial Wound Dressing.

The study reveals the polymer-crosslinker interactions and functionality of hydrophilic nanofibers for antibacterial wound coatings. Coaxial electrospinning leverages a drug encapsulation protocol for a core-shell fiber composite with a core derived from polyvinyl alcohol and polyethylene glycol with amorphous silica (PVA-PEG-SiO2), and a shell originating from polyvinyl alcohol and graphene oxide (PVA-GO). Crosslinking with GO and SiO2 initiates the hydrogel transition for the fiber composite upon contact with moisture, which aims to optimize the drug release. The effect of hydrogel-inducing additives on the drug kinetics is evaluated in the case of chlorhexidine digluconate (CHX) encapsulation in the core of core-shell fiber composite PVA-PEG-SiO2-1x-CHX@PVA-GO. The release rate is assessed with the zero, first-order, Higuchi, and Korsmeyer-Peppas kinetic models, where the inclusion of crosslinking silica provides a longer degradation and release rate. CHX medicated core-shell composite provides sustainable antibacterial activity against Staphylococcus aureus.

Two-dimensional wavefront characterization of adaptable corrective optics and Kirkpatrick-Baez mirror system using ptychography.

Aberrations introduced during fabrication degrade the performance of X-ray optics and their ability to achieve diffraction limited focusing. Corrective optics can counteract these errors by introducing wavefront perturbations prior to the optic which cancel out the distortions. Here we demonstrate two-dimensional wavefront correction of an aberrated Kirkpatrick-Baez mirror pair using adaptable refractive structures. The resulting two-dimensional wavefront is measured using hard X-ray ptychography to recover the complex probe wavefield with high spatial resolution and model the optical performance under coherent conditions. The optical performance including the beam caustic, focal profile and wavefront error is examined before and after correction with both mirrors found to be diffraction limited after correcting. The results will be applicable to a wide variety of high numerical aperture X-ray optics aiming to achieve diffraction limited focussing using low emittance sources.

A SERS platform based on diatomite modified by gold nanoparticles using a combination of layer-by-layer assembly and a freezing-induced loading method.

Siliceous diatom frustules represent an up-and-coming platform for a range of bio-assisted nanofabrication processes able to overcome the complexity and high cost of current engineering technology solutions in terms of negligibly small power consumption and environmentally friendly processing combined with unique highly porous structures and properties. Herein, the modification of diatomite - a soft, loose, and fine-grained siliceous sedimentary rock composed of the remains of fossilized diatoms - with gold nanoparticles using layer-by-layer technology in combination with a freezing-induced loading approach is demonstrated. The obtained composite structures are characterized by dynamic light scattering, extinction spectroscopy, scanning (SEM) and transmission electron microscopy (TEM), and photoacoustic imaging techniques, and tested as a platform for surface-enhanced Raman scattering (SERS) using Rhodamine 6G. SEM, TEM, and energy dispersive X-ray spectroscopy (EDX) confirmed a dense coating of gold nanoparticles with an average size of 19 nm on the surface of the diatomite and within the pores. The photoacoustic signal excited at a wavelength of 532 nm increases with increasing loading cycles of up to three polyelectrolyte-gold nanoparticle bilayers. The hybrid materials based on diatomite modified with gold nanoparticles can be used as SERS substrates, but also as biosensors, catalysts, and platforms for advanced bioimaging.

The Analysis of Micro-Scale Deformation and Fracture of Carbonized Elastomer-Based Composites by In Situ SEM.

Carbonized elastomer-based composites (CECs) possess a number of attractive features in terms of thermomechanical and electromechanical performance, durability in aggressive media and facile net-shape formability, but their relatively low ductility and strength limit their suitability for structural engineering applications. Prospective applications such as structural elements of micro-electro-mechanical systems MEMS can be envisaged since smaller principal dimensions reduce the susceptibility of components to residual stress accumulation during carbonization and to brittle fracture in general. We report the results of in situ in-SEM study of microdeformation and fracture behavior of CECs based on nitrile butadiene rubber (NBR) elastomeric matrices filled with carbon and silicon carbide. Nanostructured carbon composite materials were manufactured via compounding of elastomeric substance with carbon and SiC fillers using mixing rolling mill, vulcanization, and low-temperature carbonization. Double-edge notched tensile (DENT) specimens of vulcanized and carbonized elastomeric composites were subjected to in situ tensile testing in the chamber of the scanning electron microscope (SEM) Tescan Vega 3 using a Deben microtest 1 kN tensile stage. The series of acquired SEM images were analyzed by means of digital image correlation (DIC) using Ncorr open-source software to map the spatial distribution of strain. These maps were correlated with finite element modeling (FEM) simulations to refine the values of elastic moduli. Moreover, the elastic moduli were derived from unloading curve nanoindentation hardness measurements carried out using a NanoScan-4D tester and interpreted using the Oliver-Pharr method. Carbonization causes a significant increase of elastic moduli from 0.86 ± 0.07 GPa to 14.12 ± 1.20 GPa for the composite with graphite and carbon black fillers. Nanoindentation measurements yield somewhat lower values, namely, 0.25 ± 0.02 GPa and 9.83 ± 1.10 GPa before and after carbonization, respectively. The analysis of fractography images suggests that crack initiation, growth and propagation may occur both at the notch stress concentrator or relatively far from the notch. Possible causes of such response are discussed, namely, (1) residual stresses introduced by processing; (2) shape and size of fillers; and (3) the emanation and accumulation of gases in composites during carbonization.

Hard X-ray ptychography for optics characterization using a partially coherent synchrotron source.

Ptychography is a scanning coherent diffraction imaging technique which provides high resolution imaging and complete spatial information of the complex electric field probe and sample transmission function. Its ability to accurately determine the illumination probe has led to its use at modern synchrotrons and free-electron lasers as a wavefront-sensing technique for optics alignment, monitoring and correction. Recent developments in the ptychography reconstruction process now incorporate a modal decomposition of the illuminating probe and relax the restriction of using sources with high spatial coherence. In this article a practical implementation of hard X-ray ptychography from a partially coherent X-ray source with a large number of modes is demonstrated experimentally. A strongly diffracting Siemens star test sample is imaged using the focused beam produced by either a Fresnel zone plate or beryllium compound refractive lens. The recovered probe from each optic is back propagated in order to plot the beam caustic and determine the precise focal size and position. The power distribution of the reconstructed probe modes also allows the quantification of the beams coherence and is compared with the values predicted by a Gaussian-Schell model and the optics exit intensity.

Imaging transient melting of a nanocrystal using an X-ray laser.

There is a fundamental interest in studying photoinduced dynamics in nanoparticles and nanostructures as it provides insight into their mechanical and thermal properties out of equilibrium and during phase transitions. Nanoparticles can display significantly different properties from the bulk, which is due to the interplay between their size, morphology, crystallinity, defect concentration, and surface properties. Particularly interesting scenarios arise when nanoparticles undergo phase transitions, such as melting induced by an optical laser. Current theoretical evidence suggests that nanoparticles can undergo reversible nonhomogenous melting with the formation of a core-shell structure consisting of a liquid outer layer. To date, studies from ensembles of nanoparticles have tentatively suggested that such mechanisms are present. Here we demonstrate imaging transient melting and softening of the acoustic phonon modes of an individual gold nanocrystal, using an X-ray free electron laser. The results demonstrate that the transient melting is reversible and nonhomogenous, consistent with a core-shell model of melting. The results have implications for understanding transient processes in nanoparticles and determining their elastic properties as they undergo phase transitions.

Mitigated phase transition during first cycle of a Li-rich layered cathode studied by in operando synchrotron X-ray powder diffraction.

In operando synchrotron X-ray powder diffraction (SXPD) studies were conducted to investigate the phase transition of Li-rich Li(Li0.2Ni0.13Mn0.54Co0.13)O2 and Cr-doped Li(Li0.2Ni0.13Mn0.54Co0.03Cr0.10)O2 cathodes during the first charge/discharge cycle. Crystallographic (lattice parameters) and mechanical (domain size and microstrain) information was collected from SXPD full pattern refinement. It was found that Cr substitution at Co-site benefits in suppressing the activation of Li2MnO3 domains upon 1st charge, and thus mitigates the phase transition. As a consequence, Cr-doped layered cathode holds a better reversibility in terms of a full recovery of both lattice parameters and nano-domain size after a whole charge/discharge cycle. The effects of different cycling rates on the structural change were also discussed.

Advanced Time-Stepping Interpretation of Fly-Scan Continuous Rotation Synchrotron Tomography of Dental Enamel Demineralization.

High-resolution spatial and temporal analysis and 3D visualization of time-dependent processes, such as human dental enamel acid demineralization, often present a challenging task. Overcoming this challenge often requires the development of special methods. Dental caries remains one of the most important oral diseases that involves the demineralization of hard dental tissues as a consequence of acid production by oral bacteria. Enamel has a hierarchically organized architecture that extends down to the nanostructural level and requires high resolution to study its evolution in detail. Enamel demineralization is a dynamic process that is best investigated with the help of in situ experiments. In previous studies, synchrotron tomography was applied to study the 3D enamel structure at certain time points (time-lapse tomography). Here, another distinct approach to time-evolving tomography studies is presented, whereby the sample image is reconstructed as it undergoes continuous rotation over a virtually unlimited angular range. The resulting (single) data set contains the data for multiple (potentially overlapping) intermediate tomograms that can be extracted and analyzed as desired using time-stepping selection of data subsets from the continuous fly-scan recording. One of the advantages of this approach is that it reduces the amount of time required to collect an equivalent number of single tomograms. Another advantage is that the nominal time step between successive reconstructions can be significantly reduced. We applied this approach to the study of acidic enamel demineralization and observed the progression of demineralization over time steps significantly smaller than the total acquisition time of a single tomogram, with a voxel size smaller than 0.5 µm. It is expected that the approach presented in this paper can be useful for high-resolution studies of other dynamic processes and for assessing small structural modifications in evolving hierarchical materials.

The DIAD Approach to Correlative Synchrotron X-ray Imaging and Diffraction Analysis of Human Enamel.

The Dual Imaging and Diffraction (DIAD) beamline at Diamond Light Source (Didcot, U.K.) implements a correlative approach to the dynamic study of materials based on concurrent analysis of identical sample locations using complementary X-ray modalities to reveal structural detail at various length scales. Namely, the underlying beamline principle and its practical implementation allow the collocation of chosen regions within the sample and their interrogation using real-space imaging (radiography and tomography) and reciprocal space scattering (diffraction). The switching between the two principal modes is made smooth and rapid by design, so that the data collected is interlaced to obtain near-simultaneous multimodal characterization. Different specific photon energies are used for each mode, and the interlacing of acquisition steps allows conducting static and dynamic experiments. Building on the demonstrated realization of this state-of-the-art approach requires further refining of the experimental practice, namely, the methods for gauge volume collocation under different modes of beam-sample interaction. To address this challenge, experiments were conducted at DIAD devoted to the study of human dental enamel, a hierarchical structure composed of hydroxyapatite mineral nanocrystals, as a static sample previously affected by dental caries (tooth decay) as well as under dynamic conditions simulating the process of acid demineralization. Collocation and correlation were achieved between WAXS (wide-angle X-ray scattering), 2D (radiographic), and 3D (tomographic) imaging. While X-ray imaging in 2D or 3D modes reveals real-space details of the sample microstructure, X-ray scattering data for each gauge volume provided statistical nanoscale and ultrastructural polycrystal reciprocal-space information such as phase and preferred orientation (texture). Careful registration of the gauge volume positions recorded during the scans allowed direct covisualization of the data from two modalities. Diffraction gauge volumes were identified and visualized within the tomographic data sets, revealing the underlying local information to support the interpretation of the diffraction patterns. The present implementation of the 4D microscopy paradigm allowed following the progression of demineralization and its correlation with time-dependent WAXS pattern evolution in an approach that is transferable to other material systems.

Hierarchical modelling of elastic behaviour of human enamel based on synchrotron diffraction characterisation.

Human enamel is a hierarchical mineralized tissue with a two-level composite structure. Few studies have focused on the structure-mechanical property relationship and its link to the multi-scale architecture of human enamel, whereby the response to mechanical loading is affected not only by the rod distribution at micro-scale, but also strongly influenced by the mineral crystallite shape, and spatial arrangement and orientation. In this study, two complementary synchrotron X-ray diffraction techniques, wide and small angle X-ray scattering (WAXS/SAXS) were used to obtain multi-scale quantitative information about the structure and deformation response of human enamel to in situ uniaxial compressive loading. The apparent modulus was determined linking the external load and the internal strain in hydroxyapatite (HAp) crystallites. An improved multi-scale Eshelby model is proposed taking into account the two-level hierarchical structure of enamel. This framework has been used to analyse the experimental data for the elastic lattice strain evolution within the HAp crystals. The achieved agreement between the model prediction and experiment along the loading direction validates the model and suggests that the new multi-scale approach reasonably captures the structure-property relationship for the human enamel. The ability of the model to predict multi-directional strain components is also evaluated by comparison with the measurements. The results are useful for understanding the intricate relationship between the hierarchical structure and the mechanical properties of enamel, and for making predictions of the effect of structural alterations that may occur due to the disease or treatment on the performance of dental tissues and their artificial replacements.

A feasibility study of dynamic stress analysis inside a running internal combustion engine using synchrotron X-ray beams.

The present investigation establishes the feasibility of using synchrotron-generated X-ray beams for time-resolved in situ imaging and diffraction of the interior components of an internal combustion engine during its operation. The demonstration experiment was carried out on beamline I12 (JEEP) at Diamond Light Source, UK. The external hutch of the JEEP instrument is a large-scale engineering test bed for complex in situ processing and simulation experiments. The hutch incorporates a large capacity translation and rotation table and a selection of detectors for monochromatic and white-beam diffraction and imaging. These capabilities were used to record X-ray movies of a motorcycle internal combustion engine running at 1850 r.p.m. and to measure strain inside the connecting rod via stroboscopic X-ray diffraction measurement. The high penetrating ability and high flux of the X-ray beam at JEEP allowed the observation of inlet and outlet valve motion, as well as that of the piston, connecting rod and the timing chain within the engine. Finally, the dynamic internal strain within the moving connecting rod was evaluated with an accuracy of ~50 × 10(-6).

Hair-Reinforced Elastomer Matrix Composites: Formulation, Mechanical Testing, and Advanced Microstructural Characterization.

Epoxy matrix composites reinforced with high-performance fibers, such as carbon, Kevlar, and glass, exhibit excellent specific stiffness and strength in many mechanical applications. However, these composites are disappointingly non-recyclable and are usually disposed of in landfill sites, with no realistic prospect for biodegradation in a reasonable time. In contrast, moldable composites with carbonized elastomeric matrices developed in the last decades possess attractive mechanical properties in final net-shape products and can also be incinerated or recycled. Many carbon and inorganic fillers have recently been evaluated to adjust the properties of carbonized elastomeric composites. Renewable organic fillers, such as human or animal hair, offer an attractive fibrous material with substantial potential for reinforcing composites with elastomeric matrices. Samples of unidirectional fiber composites (with hair volume fractions up to 7%) and quasi-isotropic short fiber composites (with hair volume fractions up to 20%) of human hair-reinforced nitrile butadiene rubbers (HH-NBRs) were produced in the peroxide-cured and carbonized states. The samples were characterized using scanning electron microscopy (SEM), Raman spectroscopy, and photoacoustic microscopy. Mechanical tests were performed under tension using a miniature universal testing machine. The expected effect of fiber reinforcement on the overall mechanical performance was demonstrated for both cured and carbonized composites. Considerable enhancement of the elastic modulus (up to ten times), ultimate tensile strength (up to three times), and damage tolerance was achieved. The evidence of satisfactory interfacial bonding between hair and rubber was confirmed via SEM imaging of fracture surfaces. The suitability of photoacoustic microscopy was assessed for 3D reconstructions of the fiber sub-system's spatial distribution and non-destructive testing.

Time-Lapse In Situ 3D Imaging Analysis of Human Enamel Demineralisation Using X-ray Synchrotron Tomography.

Caries is a chronic disease that causes the alteration of the structure of dental tissues by acid dissolution (in enamel, dentine and cementum) and proteolytic degradation (dentine and cementum) and generates an important cost of care. There is a need to visualise and characterise the acid dissolution process on enamel due to its hierarchical structure leading to complex structural modifications. The process starts at the enamel surface and progresses into depth, which necessitates the study of the internal enamel structure. Artificial demineralisation is usually employed to simulate the process experimentally. In the present study, the demineralisation of human enamel was studied using surface analysis carried out with atomic force microscopy as well as 3D internal analysis using synchrotron X-ray tomography during acid exposure with repeated scans to generate a time-lapse visualisation sequence. Two-dimensional analysis from projections and virtual slices and 3D analysis of the enamel mass provided details of tissue changes at the level of the rods and inter-rod substance. In addition to the visualisation of structural modifications, the rate of dissolution was determined, which demonstrated the feasibility and usefulness of these techniques. The temporal analysis of enamel demineralisation is not limited to dissolution and can be applied to other experimental conditions for the analysis of treated enamel or remineralisation.

Synchrotron X-ray Studies of the Structural and Functional Hierarchies in Mineralised Human Dental Enamel: A State-of-the-Art Review.

Hard dental tissues possess a complex hierarchical structure that is particularly evident in enamel, the most mineralised substance in the human body. Its complex and interlinked organisation at the Ångstrom (crystal lattice), nano-, micro-, and macro-scales is the result of evolutionary optimisation for mechanical and functional performance: hardness and stiffness, fracture toughness, thermal, and chemical resistance. Understanding the physical-chemical-structural relationships at each scale requires the application of appropriately sensitive and resolving probes. Synchrotron X-ray techniques offer the possibility to progress significantly beyond the capabilities of conventional laboratory instruments, i.e., X-ray diffractometers, and electron and atomic force microscopes. The last few decades have witnessed the accumulation of results obtained from X-ray scattering (diffraction), spectroscopy (including polarisation analysis), and imaging (including ptychography and tomography). The current article presents a multi-disciplinary review of nearly 40 years of discoveries and advancements, primarily pertaining to the study of enamel and its demineralisation (caries), but also linked to the investigations of other mineralised tissues such as dentine, bone, etc. The modelling approaches informed by these observations are also overviewed. The strategic aim of the present review was to identify and evaluate prospective avenues for analysing dental tissues and developing treatments and prophylaxis for improved dental health.

Revealing the static and dynamic nanomechanical properties of diatom frustules-Nature's glass lace.

Diatoms are single cell microalgae enclosed in silica exoskeletons (frustules) that provide inspiration for advanced hybrid nanostructure designs mimicking multi-scale porosity to achieve outstanding mechanical and optical properties. Interrogating the structure and properties of diatoms down to nanometer scale leads to breakthrough advances reported here in the nanomechanical characterization of Coscinodiscus oculus-iridis diatom pure silica frustules, as well as of air-dried and wet cells with organic content. Static and dynamic mode Atomic Force Microscopy (AFM) and in-SEM nanoindentation revealed the peculiarities of diatom response with separate contributions from material nanoscale behavior and membrane deformation of the entire valve. Significant differences in the nanomechanical properties of the different frustule layers were observed. Furthermore, the deformation response depends strongly on silica hydration and on the support from the internal organic content. The cyclic loading revealed that the average compliance of the silica frustule is 0.019 m/N and increases with increasing number of cycles. The structure-mechanical properties relationship has a direct impact on the vibrational properties of the frustule as a complex micrometer-sized mechanical system. Lessons from Nature's nanostructuring of diatoms open up pathways to new generations of nano- and microdevices for electronic, electromechanical, photonic, liquid, energy storage, and other applications.

An In Vivo Rat Study of Bioresorbable Mg-2Zn-2Ga Alloy Implants.

In the present study, pins made from the novel Mg-2Zn-2Ga alloy were installed within the femoral bones of six Wistar rats. The level of bioresorption was assessed after 1, 3, and 6 months by radiography, histology, SEM, and EDX. Significant bioresorption was evident after 3 months, and complete dissolution of the pins occurred at 6 months after the installation. No pronounced gas cavities could be found at the pin installation sites throughout the postoperative period. The animals' blood parameters showed no signs of inflammation or toxication. These findings are sufficiently encouraging to motivate further research to broaden the experimental coverage to increase the number of observed animals and to conduct tests involving other, larger animals.

A concept of "materials" diffraction and imaging beamline for SKIF: Siberian circular photon source.

Over the next decade, the extremely brilliant fourth generation synchrotron radiation sources are set to become a key driving force in materials characterization and technology development. In this study, we present a conceptual design of a versatile "Materia" diffraction and imaging beamline for a low-emittance synchrotron radiation facility. The beamline was optimized for operation with three main principal delivery regimes: parallel collimated beam ∼1 mm beam size, micro-focus regime with ∼10 µm beam spot size on the sample, and nano-focus regime with <100 nm focus. All regimes will operate in the photon energy range of 10-30 keV with the key feature of the beamline being fast switching between them, as well as between the various realizations of diffraction and imaging operation modes while maintaining the target beam position at the sample, and with both spectrally narrow and spectrally broad beams up to the energy band ΔE/E of 5 × 10-2. The manuscript presents the details of the principal characteristics selected for the insertion device and beamline optics, the materials characterization techniques, including the simulations of thermal load impact on the critical beamline optics components. Significant efforts were made to design the monochromators to mitigate the very high beam power load produced by a superconducting undulator source. The manuscript will be of interest to research groups involved in the design of new synchrotron beamlines.

Revealing the origins of vortex cavitation in a Venturi tube by high speed X-ray imaging.

Hydrodynamic cavitation is useful in many processing applications, for example, in chemical reactors, water treatment and biochemical engineering. An important type of hydrodynamic cavitation that occurs in a Venturi tube is vortex cavitation known to cause luminescence whose intensity is closely related to the size and number of cavitation events. However, the mechanistic origins of bubbles constituting vortex cavitation remains unclear, although it has been concluded that the pressure fields generated by the cavitation collapse strongly depends on the bubble geometry. The common view is that vortex cavitation consists of numerous small spherical bubbles. In the present paper, aspects of vortex cavitation arising in a Venturi tube were visualized using high-speed X-ray imaging at SPring-8 and European XFEL. It was discovered that vortex cavitation in a Venturi tube consisted of angulated rather than spherical bubbles. The tangential velocity of the surface of vortex cavitation was assessed considering the Rankine vortex model.

Multi-resolution Correlative Ultrastructural and Chemical Analysis of Carious Enamel by Scanning Microscopy and Tomographic Imaging.

Caries, a major global disease associated with dental enamel demineralization, remains insufficiently understood to devise effective prevention or minimally invasive treatment. Understanding the ultrastructural changes in enamel is hampered by a lack of nanoscale characterization of the chemical spatial distributions within the dental tissue. This leads to the requirement to develop techniques based on various characterization methods. The purpose of the present study is to demonstrate the strength of analytic methods using a correlative technique on a single sample of human dental enamel as a specific case study to test the accuracy of techniques to compare regions in enamel. The science of the different techniques is integrated to genuinely study the enamel. The hierarchical structures within carious tissue were mapped using the combination of focused ion beam scanning electron microscopy with synchrotron X-ray tomography. The chemical changes were studied using scanning X-ray fluorescence (XRF) and X-ray wide-angle and small-angle scattering using a beam size below 80 nm for ångström and nanometer length scales. The analysis of XRF intensity gradients revealed subtle variations of Ca intensity in carious samples in comparison with those of normal mature enamel. In addition, the pathways for enamel rod demineralization were studied using X-ray ptychography. The results show the chemical and structural modification in carious enamel with differing locations. These results reinforce the need for multi-modal approaches to nanoscale analysis in complex hierarchically structured materials to interpret the changes of materials. The approach establishes a meticulous correlative characterization platform for the analysis of biomineralized tissues at the nanoscale, which adds confidence in the interpretation of the results and time-saving imaging techniques. The protocol demonstrated here using the dental tissue sample can be applied to other samples for statistical study and the investigation of nanoscale structural changes. The information gathered from the combination of methods could not be obtained with traditional individual techniques.