This work was supported by funding from the National Science Foundation (NSF) through grants EAR-1560779 and EAR-1647012, the Office of the VP for Research and Economic Development, the College of Arts and Sciences, and the Department of Geological Sciences. Authors also acknowledge Chiara Cappelli, Rich Martens and Johnny Goodwin for technical assistance and the Montserrat Volcano Observatory for providing the ash samples.

<ol><li>Kasama, T., Golla-Schindler, U., Putnis, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-resolution+and+energy-filtered+TEM+of+the+interface+between+hematite+and+ilmenite+exsolution+lamellae%3A+Relevance+to+the+origin+of+lamellar+magnetism%5BTitle%5D)+AND+%22American+Mineralogist%22%5BJournal%5D)">High-resolution and energy-filtered TEM of the interface between hematite and ilmenite exsolution lamellae: Relevance to the origin of lamellar magnetism.</a> American Mineralogist. 88, 1190-1196 (2003).</li>
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<li>Fougerouse, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanoscale+gold+clusters+in+arsenopyrite+controlled+by+growth+rate+not+concentration%3A+Evidence+from+atom+probe+microscopy%5BTitle%5D)+AND+%22American+Mineralogist%22%5BJournal%5D)">Nanoscale gold clusters in arsenopyrite controlled by growth rate not concentration: Evidence from atom probe microscopy.</a> American Mineralogist. 101, 1916-1919 (2016).</li>
<li>Peterman, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nanogeochronology+of+discordant+zircon+measured+by+atom+probe+microscopy+of+Pb-enriched+dislocation+loops%5BTitle%5D)+AND+%22Science+Advances%22%5BJournal%5D)">Nanogeochronology of discordant zircon measured by atom probe microscopy of Pb-enriched dislocation loops.</a> Science Advances. 2, (2016).</li>
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<li>Larson, D. J., Prosa, T. J., Ulfig, R. M., Geiser, B. P., Kelly, T. F. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aLarson%2C+DJ+%22Local+Electrode+Atom+Probe+Tomography%3A+A+User%E2%80%99s+Guide%22">Local Electrode Atom Probe Tomography: A User’s Guide</a>. , Springer. New York. 318(2013).</li>
<li>Devine, J. D., Rutherford, M. J., Norton, G. E., Young, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Magma+storage+region+processes+inferred+from+geochemistry+of+Fe%E2%80%93Ti+oxides+in+andesitic+magma%2C+Soufriere+Hills+Volcano%5BTitle%5D)+AND+%22Journal+of+Petrology%22%5BJournal%5D)">Magma storage region processes inferred from geochemistry of Fe–Ti oxides in andesitic magma, Soufriere Hills Volcano.</a> Journal of Petrology. 44, Montserrat, WI. 1375-1400 (2003).</li>
<li>Jackson, M., Bowles, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Curie+temperatures+of+titanomagnetite+in+ignimbrites%3A+Effects+of+emplacement+temperatures%2C+cooling+rates%2C+exsolution%2C+and+cation+ordering%5BTitle%5D)+AND+%22Geochemistry%2C+Geophysics%2C+Geosystems%22%5BJournal%5D)">Curie temperatures of titanomagnetite in ignimbrites: Effects of emplacement temperatures, cooling rates, exsolution, and cation ordering.</a> Geochemistry, Geophysics, Geosystems. 15, 4343-4368 (2014).</li>
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</ol>

The authors have nothing to disclose.

3D APT data reconstructions allow a precise measurement of the interlamellar spacing in the analyzed crystal at a resolution three orders of magnitude higher than those measured from conventional SEM images. This indicates that atomic variations in chemistry occur over a spatial extent three orders of magnitude smaller than optically observable mineralogical changes. Also, the measured interlamellar distances (29 nm and 14 nm), are consistent with the length scale for oxyexsolution as opposed to that for nucleation and g...

The study of chemical mineralogy has been a major source of information within the field of Earth Sciences for more than a century, as minerals actively record geological processes during and after their crystallization. Physio-chemical conditions of these processes, such as temperature changes during volcanism and metamorphism, are recorded during mineral nucleation and growth in the form of chemical zonation, striations, and lamellae, among others. Exsolution lamellae form when a phase unmixes into two separate phases in the solid state. The analysis of the orientation, size, morphology, and spacing of such exsolution lamellae can provide essential information to understand temperature and pressure changes during volcanism and metamorphism1,2,3 and the formation of ore mineral deposits4.
Traditionally, the study of exsolution lamellae was conducted with the observation of micrographs by simple scanning electron imaging5. More recently, this has been substituted by the use of energy-filtered transmission electron microscopy (TEM) providing detailed observations at the nanoscale level1,2,3. Nevertheless, in both cases, the observations are made in two dimensions (2D), which is not fully adequate for three dimensional (3D) structures represented by these exsolution lamellae. Nanotomography6 is emerging as a new technique for the 3D observation of nanoscale features inside minerals grains, yet there is insufficient information about the composition of these features. An alternative to these approaches is the use of atom probe tomography (APT), representing the highest spatial resolution analytical technique in existence for the characterization of materials7. The strength of the technique lies in the possibility of combining a 3D reconstruction of nanoscale features with their chemical composition at the atomic scale with a near part-per-million analytical sensitivity7. Previous applications of APT to the analysis of geological samples have provided excellent results8,9,10,11, particularly in the chemical characterization of element diffusion and concentrations9,12,13. Yet, this application has not been used for the study of exsolution lamellae, abundant in some minerals hosted in metamorphic and igneous rocks. Here, we explore the use of APT, and its limitations, for the analysis of the size and composition of exsolution lamellae, and interlamellar spacing in volcanic titanomagnetite crystals.

<table><tbody><tr><td>InTouchScope Secondary Electron Microscope (SEM)</td><td>JEOL</td><td>JSM-6010PLUS/LA</td><td></td></tr><tr><td>Focus Ion Beam (FIB) Secondary Electron Microscope (SEM)</td><td>TESCAN</td><td>LYRA XMU</td><td></td></tr><tr><td>Local Electrode Atom Probe (LEAP)</td><td>CAMECA</td><td>5000 XS</td><td></td></tr><tr><td>Integrated Visualization and Analysis Software (IVAS, version 3.6.12).</td><td></td><td></td><td>processing software</td></tr></tbody></table>

atom probe tomography analysis of exsolved mineral phases

Element diffusion rates and temperature/pressure control a range of fundamental volcanic and metamorphic processes. Such processes are often recorded in lamellae exsolved from host mineral phases. Thus, the analysis of the orientation, size, morphology, composition and spacing of exsolution lamellae is an area of active research in the geosciences. The conventional study of these lamellae has been conducted by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and more recently with focused ion beam (FIB)-based nanotomography, yet with limited chemical information. Here, we explore the use of atom probe tomography (APT) for the nanoscale analysis of ilmenite exsolution lamellae in igneous titanomagnetite from ash deposits erupted from the active Soufri&#232;re Hills Volcano (Montserrat, British West Indies). APT allows the precise calculation of interlamellar spacings (14&#8211;29 &#177; 2 nm) and reveals smooth diffusion profiles with no sharp phase boundaries during the exchange of Fe and Ti/O between the exsolved lamellae and the host crystal. Our results suggest that this novel approach permits nanoscale measurements of lamellae composition and interlamellar spacing that may provide a means to estimate the lava dome temperatures necessary to model extrusion rates and lava dome failure, both of which play a key role in volcanic hazard mitigation efforts.

Like many titanomagnetite crystals from various stages of the Soufri&#232;re Hills Volcano (SHV) eruption, the crystal analyzed here contains exsolution lamellae &#60;10 &#181;m in thickness, visible in secondary SEM images (Figure 1d), which separate zones of Ti-rich magnetite, indicating a C2 stage of oxidation18. Based upon the SEM images, spacing between these lamellae ranges from 2 to 6 &#181;m (n = 15). Four titanomagnetite specimen tips, referred as 20...

Watch this Scientific Journal Video about Atom Probe Tomography Analysis of Exsolved Mineral Phases at JoVE.com

Atom Probe Tomography Analysis of Exsolved Mineral Phases

Analysis of the morphology, composition, and spacing of exsolution lamellae can provide essential information to understand geological processes related to volcanism and metamorphism. We present a novel application of APT for the characterization of such lamellae and compare this approach to the conventional use of electron microscopy and FIB-based nanotomography.

Element diffusion rates and temperature/pressure control a range of fundamental volcanic and metamorphic processes. Such processes are often recorded in ...

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Environment

7.3K Views.  University of Alabama. Het onderzoeken van variaties in minerale chemie kan licht werpen op veranderingen in vulkanische activiteit, en onderzoekers in staat stellen om tijdschema's van vulkanische processen te verkrijgen om eventuele gevaren beter te begrijpen. Atoomsonde tomografie maakt een ongekende 3D-visualisatie van minerale exsolved fasen mogelijk terwijl ze hun chemische samenstelling op atomaire schaal meten. We passen de techniek momenteel toe op de karakterisering van pathologische mineralisatie, bijvoo...

Video: Atom Probe Tomography Analysis of Exsolved Mineral Phases

Atom Probe Tomography Studies on the Cu(In,Ga)Se2 Grain Boundaries

Compared with the existent techniques, atom probe tomography is a unique technique able to chemically characterize the internal interfaces at the nanoscale and in three dimensions. Indeed, APT possesses high sensitivity (in the order of ppm) and high spatial resolution (sub nm).
Considerable efforts were done here to prepare an APT tip which contains the desired grain boundary with a known structure. Indeed, site-specific sample preparation using combined focused-ion-beam, electron backscatter diffraction, and transmission electron microscopy is presented in this work. This method allows selected grain boundaries with a known structure and location in Cu(In,Ga)Se2 thin-films to be studied by atom probe tomography.
Finally, we discuss the advantages and drawbacks of using the atom probe tomography technique to study the grain boundaries in Cu(In,Ga)Se2 thin-film solar cells.

Atom Probe Tomography Studies on the CuIn,GaSe2 Grain Boundaries

In this work, we describe the use of the atom-probe tomography technique for studying the grain boundaries of the absorber layer in a CIGS solar cell. A novel approach to prepare the atom probe tips containing the desired grain boundary with a known structure is also presented here.

Compared with the existent techniques, atom probe tomography is a unique technique able to chemically characterize the internal interfaces at the ...

Engineering

Reservoir Condition Pore-scale Imaging of Multiple Fluid Phases Using X-ray Microtomography

X-ray microtomography was used to image, at a resolution of 6.6 &#181;m, the pore-scale arrangement of residual carbon dioxide ganglia in the pore-space of a carbonate rock at pressures and temperatures representative of typical formations used for CO2 storage. Chemical equilibrium between the CO2, brine and rock phases was maintained using a high pressure high temperature reactor, replicating conditions far away from the injection site. Fluid flow was controlled using high pressure high temperature syringe pumps. To maintain representative in-situ conditions within the micro-CT scanner a carbon fiber high pressure micro-CT coreholder was used. Diffusive CO2 exchange across the confining sleeve from the pore-space of the rock to the confining fluid was prevented by surrounding the core with a triple wrap of aluminum foil. Reconstructed brine contrast was modeled using a polychromatic x-ray source, and brine composition was chosen to maximize the three phase contrast between the two fluids and the rock. Flexible flow lines were used to reduce forces on the sample during image acquisition, potentially causing unwanted sample motion, a major shortcoming in previous techniques. An internal thermocouple, placed directly adjacent to the rock core, coupled with an external flexible heating wrap and a PID controller was used to maintain a constant temperature within the flow cell. Substantial amounts of CO2 were trapped, with a residual saturation of 0.203 &#177; 0.013, and the sizes of larger volume ganglia obey power law distributions, consistent with percolation theory.

We present a methodology for the imaging of multiple fluid phases at reservoir conditions by the use of x-ray microtomography. We show some representative results of capillary trapping in a carbonate rock sample.

X-ray microtomography was used to image, at a resolution of 6.6 &#181;m, the pore-scale arrangement of residual carbon dioxide ganglia in the pore-space ...

Energy Dispersive X-ray Tomography for 3D Elemental Mapping of Individual Nanoparticles

Energy dispersive X-ray spectroscopy within the scanning transmission electron microscope (STEM) provides accurate elemental analysis with high spatial resolution, and is even capable of providing atomically resolved elemental maps. In this technique, a highly focused electron beam is incident upon a thin sample and the energy of emitted X-rays is measured in order to determine the atomic species of material within the beam path. This elementally sensitive spectroscopy technique can be extended to three dimensional tomographic imaging by acquiring multiple spectrum images with the sample tilted along an axis perpendicular to the electron beam direction.
Elemental distributions within single nanoparticles are often important for determining their optical, catalytic and magnetic properties. Techniques such as X-ray tomography and slice and view energy dispersive X-ray mapping in the scanning electron microscope provide elementally sensitive three dimensional imaging but are typically limited to spatial resolutions of &gt; 20 nm. Atom probe tomography provides near atomic resolution but preparing nanoparticle samples for atom probe analysis is often challenging. Thus, elementally sensitive techniques applied within the scanning transmission electron microscope are uniquely placed to study elemental distributions within nanoparticles of dimensions 10-100 nm.
Here, energy dispersive X-ray (EDX) spectroscopy within the STEM is applied to investigate the distribution of elements in single AgAu nanoparticles. The surface segregation of both Ag and Au, at different nanoparticle compositions, has been observed.

The use of energy dispersive X-ray tomography in the scanning transmission electron microscope to characterize elemental distributions within single nanoparticles in three dimensions is described.

Energy dispersive X-ray spectroscopy within the scanning transmission electron microscope (STEM) provides accurate elemental analysis with high spatial ...

Dynamic Pore-scale Reservoir-condition Imaging of Reaction in Carbonates Using Synchrotron Fast Tomography

Underground storage permanence is a major concern for carbon capture and storage. Pumping CO2 into carbonate reservoirs has the potential to dissolve geologic seals and allow CO2 to escape. However, the dissolution processes at reservoir conditions are poorly understood. Thus, time-resolved experiments are needed to observe and predict the nature and rate of dissolution at the pore scale. Synchrotron fast tomography is a method of taking high-resolution time-resolved images of complex pore structures much more quickly than traditional &#181;-CT. The Diamond Lightsource Pink Beam was used to dynamically image dissolution of limestone in the presence of CO2-saturated brine at reservoir conditions. 100 scans were taken at a 6.1 &#181;m resolution over a period of 2 hours. The images were segmented and the porosity and permeability were measured using image analysis and network extraction. Porosity increased uniformly along the length of the sample; however, the rate of increase of both porosity and permeability slowed at later times.

Synchrotron fast tomography was used to dynamically image dissolution of limestone in the presence of CO2-saturated brine at reservoir conditions. 100 scans were taken at a 6.1 &#181;m resolution over a period of 2 h.

Underground storage permanence is a major concern for carbon capture and storage. Pumping CO2 into carbonate reservoirs has the potential to dissolve ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Bioengineering

Processing of Bulk Nanocrystalline Metals at the US Army Research Laboratory

Given their potential for significant property improvements relative to their large grained counterparts, much work has been devoted to the continued development of nanocrystalline metals. Despite these efforts, the transition of these materials from the lab bench to actual applications has been blocked by the inability to produce large scale parts that retain the desired nanocrystalline microstructures. Following the development of a method proven to stabilize the nanosized grain structure to temperatures approaching that of the melting point for the given metal, the US Army Research Laboratory (ARL) has progressed to the next stage in the development of these materials - namely the production of large scale parts suitable for testing and evaluation in a range of relevant test environments. This report provides a broad overview of the ongoing efforts in the processing, characterization, and consolidation of these materials at ARL. In particular, focus is placed on the methodology used for producing the nanocrystalline metal powders, in both small and large-scale amounts, that are at the center of ongoing research efforts.

This paper provides a brief overview of the ongoing efforts at the Army Research Laboratory on the processing of bulk nanocrystalline metals with an emphasis on the methodologies used for the production of the novel metal powders.

Given their potential for significant property improvements relative to their large grained counterparts, much work has been devoted to the continued ...

Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. These cell lines, after treatment with ascorbic acid (AA) and &#946;-glycerophosphate (&#946;-GP), undergo complete osteogenic transdifferentiation from proliferation to mineralization and produce matrix vesicles (MVs) that trigger apatite nucleation in the extracellular matrix (ECM).
Based on Alizarin Red-S (AR-S) staining and analysis of the composition of minerals in cell lysates using ultraviolet (UV) light or in vesicles using TEM imaging followed by EDX quantitation and ion mapping, we can infer that osteosarcoma Saos-2 and osteoblastic hFOB 1.19 cells reveal distinct mineralization profiles. Saos-2 cells mineralize more efficiently than hFOB 1.19 cells and produce larger mineral deposits that are not visible under UV light but are similar to hydroxyapatite (HA) in that they have more Ca and F substitutions.
The results obtained using these techniques allow us to conclude that the process of mineralization differs depending on the cell type. We propose that, at the cellular level, the origin and properties of vesicles predetermine the type of minerals.

We present a protocol to compare the state of minerals in vesicles released by two human bone cell lines: hFOB 1.19 and Saos-2. Their mineralization profiles were analyzed by Alizarin Red-S (AR-S) staining, ultraviolet (UV) light visualization, transmission electron microscopy (TEM) imaging and energy dispersive X-ray microanalysis (EDX).

This video presents the use of transmission electron microscopy with energy dispersive X-ray microanalysis (TEM-EDX) to compare the state of minerals in ...

Chemistry

Pore-scale Imaging and Characterization of Hydrocarbon Reservoir Rock Wettability at Subsurface Conditions Using X-ray Microtomography

In situ wettability measurements in hydrocarbon reservoir rocks have only been possible recently. The purpose of this work is to present a protocol to characterize the complex wetting conditions of hydrocarbon reservoir rock using pore-scale three-dimensional X-ray imaging at subsurface conditions. In this work, heterogeneous carbonate reservoir rocks, extracted from a very large producing oil field, have been used to demonstrate the protocol. The rocks are saturated with brine and oil and aged over three weeks at subsurface conditions to replicate the wettability conditions that typically exist in hydrocarbon reservoirs (known as mixed-wettability). After the brine injection, high-resolution three-dimensional images (2 &#181;m/voxel) are acquired and then processed and segmented. To calculate the distribution of the contact angle, which defines the wettability, the following steps are performed. First, fluid-fluid and fluid-rock surfaces are meshed. The surfaces are smoothed to remove voxel artefacts, and in situ contact angles are measured at the three-phase contact line throughout the whole image. The main advantage of this method is its ability to characterize in situ wettability accounting for pore-scale rock properties, such as rock surface roughness, rock chemical composition, and pore size. The in situ wettability is determined rapidly at hundreds of thousands of points. 
The method is limited by the segmentation accuracy and X-ray image resolution. This protocol could be used to characterize the wettability of other complex rocks saturated with different fluids and at different conditions for a variety of applications. For example, it could help in determining the optimal wettability that could yield an extra oil recovery (i.e., designing brine salinity accordingly to obtain higher oil recovery) and to find the most efficient wetting conditions to trap more CO2 in subsurface formations.

This protocol is presented to characterize the complex wetting conditions of an opaque porous medium (hydrocarbon reservoir rock) using three-dimensional images obtained by X-ray microtomography at subsurface conditions.

In situ wettability measurements in hydrocarbon reservoir rocks have only been possible recently. The purpose of this work is to present a protocol to ...

Electron Cryotomography of Bacterial Cells

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for instance how they generate and maintain specific cell shapes, establish polarity, segregate their genomes, and divide. In order to understand these phenomena, imaging technologies are needed that bridge the resolution gap between fluorescence light microscopy and higher-resolution methods such as X-ray crystallography and NMR spectroscopy.
Electron cryotomography (ECT) is an emerging technology that does just this, allowing the ultrastructure of cells to be visualized in a near-native state, in three dimensions (3D), with "macromolecular" resolution (~4nm).1, 2 In ECT, cells are imaged in a vitreous, "frozen-hydrated" state in a cryo transmission electron microscope (cryoTEM) at low temperature (&lt; -180&deg;C). For slender cells (up to ~500 nm in thickness3), intact cells are plunge-frozen within media across EM grids in cryogens such as ethane or ethane/propane mixtures. Thicker cells and biofilms can also be imaged in a vitreous state by first "high-pressure freezing" and then, "cryo-sectioning" them. A series of two-dimensional projection images are then collected through the sample as it is incrementally tilted along one or two axes. A three-dimensional reconstruction, or "tomogram" can then be calculated from the images. While ECT requires expensive instrumentation, in recent years, it has been used in a few labs to reveal the structures of various external appendages, the structures of different cell envelopes, the positions and structures of cytoskeletal filaments, and the locations and architectures of large macromolecular assemblies such as flagellar motors, internal compartments and chemoreceptor arrays.1, 2
In this video article we illustrate how to image cells with ECT, including the processes of sample preparation, data collection, tomogram reconstruction, and interpretation of the results through segmentation and in some cases correlation with light microscopy.

We illustrate here how to use electron cryotomography (ECT) to study the ultrastructure of bacterial cells in near-native states, to "macromolecular" (~4 nm) resolution.

While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for ...

Biology

Multimodal Analysis of Bone Formation Using Explant Models

Comprehensive Characterization of Tissue Mineralization in an Ex Vivo Model

The extensive characterization of tissue mineralization in the context of bone regeneration represents a significant challenge, given the numerous modalities that are currently available for analysis. Here, we propose a workflow for a comprehensive evaluation of new bone formation using a relevant large animal osseous ex vivo explant. A bone defect (diameter = 3.75 mm; depth = 5.0 mm) is created in an explanted sheep femoral head and injected with a macroporous bone substitute loaded with a pro-osteogenic growth factor (bone morphogenetic protein 2 - BMP2). Subsequently, the explant is maintained in culture for a 28-day period, allowing cellular colonization and subsequent bone formation. To evaluate the quality and structure of newly mineralized tissue, the following successive methods are set up: (i) Characterization and high-resolution 3D images of the entire explant using micro-CT, followed by deep learning image analyses to enhance the discrimination of mineralized tissues; (ii) Nano-indentation to determine the mechanical properties of the newly formed tissue; (iii) Histological examinations, such as Hematoxylin/Eosin/Saffron (HES), Goldner's trichrome, and Movat's pentachrome to provide a qualitative assessment of mineralized tissue, particularly with regard to the visualization of the osteoid barrier and the presence of bone cells; (iv) Back-scattering scanning electron microscopy (SEM) mapping with internal reference to quantify the degree of mineralization and provide detailed insights into surface morphology, mineral composition, and bone-biomaterial interface; (v) Raman spectroscopy to characterize the molecular composition of the mineralized matrix and to provide insights into the persistence of BMP2 within the cement through the detection of peptide bonds. This multimodal analysis will provide an effective assessment of newly formed bone and comprehensive qualitative and quantitative insights into mineralized tissues. Through the standardization of these protocols, we aim to facilitate interstudy comparisons and improve the validity and reliability of research findings.

Author Spotlight: Advanced Techniques for Characterizing Tissue Mineralization in Bone Regeneration Research

The proposed protocol entails a global approach to assess bone formation in the context of bone regeneration using multimodal analyses. It aims to provide qualitative and quantitative information on new bone formation, enhancing the rigor and validity of basic and pre-clinical investigations.

Atom Probe Tomography Analysis of Exsolved Mineral Phases

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