Publications

We employed in a correlative manner an unconventional combination of methods, comprising cathodoluminescence, cryo–scanning electron microscopy (SEM), and cryo–focused ion beam (FIB)-SEM, to examine the volumes of thousands of cubed micrometers from rabbit atherosclerotic tissues, maintained in close-to-native conditions, with a resolution of tens of nanometers. Data from three different intralesional regions, at the media–lesion interface, in the core, and toward the lumen, were analyzed following segmentation and volume or surface representation. The media–lesion interface region is rich in cells and lipid droplets, whereas the core region is markedly richer in crystals and has lower cell density. In the three regions, thin crystals appear to be associated with intracellular or extracellular lipid droplets and multilamellar bodies. Large crystals are independently positioned in the tissue, not associated with specific cellular components. This extensive evidence strongly supports the idea that the lipid droplet surfaces and the outer membranes of multilamellar bodies play a role in cholesterol crystal nucleation and growth and that crystal formation occurs, in part, inside cells. The correlative combination of methods that allowed the direct examination of cholesterol crystals and lipid deposits in the atherosclerotic lesions may be similarly used for high-resolution examination of other tissues containing pathological or physiological cholesterol deposits.

We revisit the important issues of polymorphism, structure, and nucleation of cholesterol·H2O using first-principles calculations based on dispersion-augmented density functional theory. For the lesser known monoclinic polymorph, we obtain a fully extended H-bonded network in a structure akin to that of hexagonal ice. We show that the energy of the monoclinic and triclinic polymorphs is similar, strongly suggesting that kinetic and environmental effects play a significant role in determining polymorph nucleation. Furthermore, we find evidence in support of various O–H···O bonding motifs in both polymorphs that may result in hydroxyl disorder. We have been able to explain, via computation, why a single cholesterol bilayer in hydrated membranes always crystallizes in the monoclinic polymorph. We rationalize what we believe is a single-crystal to single-crystal transformation of the monoclinic form on increased interlayer growth beyond that of a single cholesterol bilayer, interleaved by a water bilayer. We show that the ice-like structure is also relevant to the related cholestanol·2H2O and stigmasterol·H2O crystals. The structure of stigmasterol hydrate both as a trilayer film at the air–water interface and as a macroscopic crystal further assists us in understanding the polymorphic and thermal behavior of cholesterol·H2O. Finally, we posit a possible role for one of the sterol esters in the crystallization of cholesterol·H2O in pathological environments, based on a composite of a crystalline bilayer of cholesteryl palmitate bound epitaxially as a nucleating agent to the monoclinic cholesterol·H2O form.

The interphase region at the base of the growth plate includes blood vessels, cells and mineralized tissues. In this region, cartilage is mineralized and replaced with bone. Blood vessel extremities permeate this space providing nutrients, oxygen and signaling factors. All these different components form a complex intertwined 3D structure. Here we use cryo-FIB SEM to elaborate this 3D structure without removing the water. As it is challenging to image mineralized and unmineralized tissues in a hydrated state, we provide technical details of the parameters used. We obtained two FIB SEM image stacks that show that the blood vessels are in intimate contact not only with cells, but in some locations also with mineralized tissues. There are abundant red blood cells at the extremities of the vessels. We also documented large multinucleated cells in contact with mineralized cartilage and possibly also with bone. We observed membrane bound mineralized particles in these cells, as well as in blood serum, but not in the hypertrophic chondrocytes. We confirm that there is an open pathway from the blood vessel extremities to the mineralizing cartilage. Based on the sparsity of the mineralized particles, we conclude that mainly ions in solution are used for mineralizing cartilage and bone, but these are augmented by the supply of mineralized particles.

Reflective assemblies of high refractive index organic crystals are used to produce striking optical phenomena in organisms based on light reflection and scattering. In aquatic animals, organic crystal-based reflectors are used both for image-formation and to increase photon capture. Here we report the characterization of a poorly-documented reflector in the eye of the shrimp L. vannamei lying 150 μm below the retina, which we term the proximal reflective layer (PR-layer). The PR-layer is made from a dense but disordered array of polycrystalline isoxanthopterin nanoparticles, similar to those recently reported in the tapetum of the same animal. Each spherical nanoparticle is composed of numerous isoxanthopterin single crystal plates arranged in concentric lamellae around an aqueous core. The highly reflective plate faces of the crystals are all aligned tangentially to the particle surface with the optical axes projecting radially outwards, forming a birefringent spherulite which efficiently scatters light. The nanoparticle assemblies form a broadband reflective sheath around the screening pigments of the eye, resulting in pronounced eye-shine when the animal is viewed from a dorsal-posterior direction, rendering the eye pigments inconspicuous. We assess possible functions of the PR-layer and conclude that it likely functions as a camouflage device to conceal the dark eye pigments in an otherwise largely transparent animal.

The birefringence of isoxanthopterin crystalline spherulites enhances the reflectivity of a biological photonic crystal.Spectacular natural optical phenomena are produced by highly reflective assemblies of organic crystals. Here we show how the tapetum reflector in a shrimp eye is constructed from arrays of spherical isoxanthopterin nanoparticles and relate the particle properties to their optical function. The nanoparticles are composed of single-crystal isoxanthopterin nanoplates arranged in concentric lamellae around a hollow core. The spherulitic birefringence of the nanoparticles, which originates from the radial alignment of the plates, results in a significant enhancement of the back-scattering. This enables the organism to maximize the reflectivity of the ultrathin tapetum, which functions to increase the eye's sensitivity and preserve visual acuity. The particle size, core/shell ratio and packing are also controlled to optimize the intensity and spectral properties of the tapetum back-scattering. This system offers inspiration for the design of photonic crystals constructed from spherically symmetric birefringent particles for use in ultrathin reflectors and as non-iridescent pigments.

Guanine crystals are used by certain animals, including vertebrates, to produce structural colors or to enhance vision, because of their distinctive reflective properties. Here we use cryo-SEM, cryo-FIB SEM and Raman spectroscopic imaging to characterize crystalline inclusions in a single celled photosynthesizing marine dinoflagellate species. We demonstrate spectroscopically that these inclusions are blocky crystals of anhydrous guanine in the beta-polymorph. Two-dimensional cryo-SEM and three-dimensional cryo-FIB-SEM serial block face imaging show that the deposits of anhydrous guanine crystals are closely associated with the chloroplasts. We suggest that the crystalline deposits scatter light either to enhance light exploitation by the chloroplasts, or possibly for protection from UV radiation. This is consistent with the crystal locations within the cell, their shapes and their sizes. As the dinoflagellates are extremely abundant in the oceans and are a major group of photosynthesizing marine organisms, the presence of guanine crystals in this marine organism may have broad significance.

Shelled pteropods are holoplanktonic mollusks that build lightweight shells containing aragonite crystals. The complex shell architecture is composed of well-aligned, curved aragonitic fibers. Each curved fiber is continuously crystalline. We used in vivo micro-Raman spectroscopy to study the mineral composition of shells of living Creseis acicula pteropods at the larval (veliger) and adult stages. The spectra obtained from the growing edge have weak and broad peaks indicative of a highly disordered nascent aragonite phase. The disordered precursor phase is detected in the newly formed regions both at the shell edge, and during thickening in the internal part of the shell. As the shell grows and thickens throughout the life of the animal, the mineral matures from a disordered transient precursor phase to crystalline aragonite. We conclude that the shell of C. acicula is formed via a disordered nascent form of aragonite, which, being isotropic, facilitates the formation of the convoluted morphology in the continuously crystalline fibers of aragonite.

We investigate the structures and mechanical properties of leaf midribs of Ficus microcarpa and Prunus dulcis, which deposit calcium oxalate crystals, and of Olea europaea midribs which contain no mineral deposits, but do contain lignified fibers. The midrib mechanical performance contributes to the leafs ability to maintain a flat conformation for light harvesting and to efficiently reconfigure to reduce wind drag. We use a novel approach involving 3D visualization of the vein structure during mechanical load. This involves the use of customized mechanical loading devices that fit inside a microCT chamber. We show that the elastic, compression and torsional moduli of the midribs of leaves from the 3 species examined vary significantly. We also observed different modes of fracture and buckling of the leaves during compression. We assess the contributions of the calcium oxalate crystals to the mechanical and fracture properties. In F. microcarpa midrib linear arrays of calcium oxalate crystals contribute to resisting the bending, in contrast to P. dulcis leaves, where the calcium oxalate crystals do not resist bending. In both F. microcarpa and P. dulcis isolated calcium oxalate crystals enable high torsional compliance. The integrated microCT- mechanical testing approach could be used to investigate the structure-mechanics relationships in other complex biological samples.Statement of significanceLeaves need to maintain a flat conformation for light harvesting, but they also need to efficiently reconfigure to reduce wind drag. The leaf central vein (midrib) is a key structural component for leaf mechanicss. 3D visualization of the vein structure under mechanical loads showed that veins can be stiffened by reinforcement units composed of calcium oxalates crystals and lignin. The stiffening units can influence the bending and fracture properties of the midribs, and can contribute to determine if buckling will occur during folding. Mineral stiffening elements could be a widespread strategy to reinforce leaf veins and other biological structures. This structural-mechanical approach could be used to study other complex biological samples. (C) 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Quantifying ion concentrations and mapping their intracellular distributions at high resolution can provide much insight into the formation of biomaterials. The key to achieving this goal is cryo-fixation, where the biological materials, tissues and associated solutions are rapidly frozen and preserved in a vitreous state. We developed a correlative cryo-Scanning Electron Microscopy (SEM)/Energy Dispersive Spectroscopy (EDS) protocol that provides quantitative elemental analysis correlated with spatial imaging of cryo-immobilized specimens. We report the accuracy and sensitivity of the cryo-EDS method, as well as insights we derive on biomineralization pathways in a foraminifer. Foraminifera are marine protozoans that produce Mg-containing calcitic shells and are major calcifying organisms in the oceans. We use the cryo-SEM/EDS correlative method to characterize unusual Mg and Ca-rich particles in the cytoplasm of a benthic foraminifer. The Mg/Ca ratio of these particles is consistently lower than that of seawater, the source solution for these ions. We infer that these particles are involved in Ca ion supply to the shell. We document the internal structure of the MgCa particles, which in some cases include a separate Si rich core phase. This approach to mapping ion distribution in cryo-preserved specimens may have broad applications to other mineralized biomaterials.Statement of significanceIons are an integral part of life, and some ions play fundamental roles in cell metabolism. Determining the concentrations of ions in cells and between cells, as well as their distributions at high resolution can provide valuable insights into ion uptake, storage, functions and the formation of biomaterials. Here we present a new cryo-SEM/EDS protocol that allows the mapping of different ion distributions in solutions and biological samples that have been cryo-preserved. We demonstrate the value of this novel approach by characterizing a novel biogenic mineral phase rich in Mg found in foraminifera, single celled marine organisms. This method has wide applicability in biology, and especially in understanding the formation and function of mineral-containing hard tissues. (C) 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The formation of atherosclerotic plaques in the blood vessel walls is the result of LDL particle uptake, and consequently of cholesterol accumulation in macrophage cells. Excess cholesterol accumulation eventually results in cholesterol crystal deposition, the hallmark of mature atheromas. We followed the formation of cholesterol crystals in J774A.1 macrophage cells with time, during accumulation of LDL particles, using a previously developed correlative cryosoft X-ray tomography (cryo-SXT) and stochastic optical reconstruction microscopy (STORM) technique. We show, in the initial accumulation stages, formation of small quadrilateral crystal plates associated with the cell plasma membrane, which may subsequently assemble into large aggregates. These plates match crystals of the commonly observed cholesterol monohydrate triclinic structure. Large rod-like cholesterol crystals form at a later stage in intracellular locations. Using cryotransmission electron microscopy (cryo-TEM) and cryoelectron diffraction (cryo-ED), we show that the structure of the large elongated rods corresponds to that of monoclinic cholesterol monohydrate, a recently determined polymorph of the triclinic crystal structure. These monoclinic crystals form with an unusual hollow cylinder or helical architecture, which is preserved in the mature rodlike crystals. The rod-like morphology is akin to that observed in crystals isolated from atheromas. We suggest that the crystals in the atherosclerotic plaques preserve in their morphology the memory of the structure in which they were formed. The identification of the polymorph structure, besides explaining the different crystal morphologies, may serve to elucidate mechanisms of cholesterol segregation and precipitation in atherosclerotic plaques.

Scallops possess a visual system comprising up to 200 eyes, each containing a concave mirror rather than a lens to focus light. The hierarchical organization of the multilayered mirror is controlled for image formation, from the component guanine crystals at the nanoscale to the complex three-dimensional morphology at the millimeter level. The layered structure of the mirror is tuned to reflect the wavelengths of light penetrating the scallop's habitat and is tiled with a mosaic of square guanine crystals, which reduces optical aberrations. The mirror forms images on a double-layered retina used for separately imaging the peripheral and central fields of view. The tiled, off-axis mirror of the scallop eye bears a striking resemblance to the segmented mirrors of reflecting telescopes.

Fish-scale iridophore cells deposit guanine crystals and assemble them into multilayer reflectors to produce silvery reflectance. The crystal orientation controls the reflective properties of the fish scales, but little is known about the degree of orientation of the guanine crystals and whether this orientation is pre-determined at the level of an individual cell. Koi fish-scale-attached iridophores, iridophores on regenerated scales, and cultured iridophores were examined by using light microscopy and synchrotron micro-X-ray diffraction. More than 95% of the thin {100} guanine crystal plates in the iridophores of the mature and regenerated scales are oriented parallel to the scale surface and perpendicular to the direction of the incoming light. More than 70% of the crystals in cultured iridophore cells are also in this orientation. The crystals are elongated and within each cell on the mature scale and in the cultured cells the long morphological axes are well aligned with the long axis of the iridophore. In contrast to the cultured iridophores, in the mature scale the iridophore cells are co-aligned with each other. Cultured iridophores are flexible and motile, and azimuthal crystal orientations vary as the cells move. We conclude that iridophore cells function as independent units and that the control over crystal orientation is pre-determined at the individual cell level in the direction that is essential for function, namely, exposing the large reflecting crystal surface to light.

Bone homeostasis is continuously regulated by the coordinated action of bone-resorbing osteoclasts and bone-forming osteoblasts. Imbalance between these two cell populations leads to pathological bone diseases such as osteoporosis and osteopetrosis. Osteoclast functionality relies on the formation of sealing zone (SZ) rings that define the resorption lacuna. It is commonly assumed that the structure and dynamic properties of the SZ depend on the physical and chemical properties of the substrate. Considering the unique complex structure of native bone, elucidation of the relevant parameters affecting SZ formation and stability is challenging. In this study, we examined in detail the dynamic response of the SZ to the microtopography of devitalized bone surfaces, taken from the same area in cattle femur. We show that there is a significant enrichment in large and stable SZs (diameter larger than 14 mu m; lifespan of hours) in cells cultured on rough bone surfaces, compared with small and fast turning over SZ rings (diameter below 7 mu m; lifespan approx. 7 min) formed on smooth bone surfaces. Based on these results, we propose that the surface roughness of the physiologically relevant substrate of osteoclasts, namely bone, affects primarily the local stability of growing SZs.

The Rosarium philosophorum, published in Frankfurt in 1550, is an anonymous text of alchemy. The text constantly shifts between physical alchemy, statements about experiments with substance, and the realm of soul alchemy, the task of the inner transmutation of forces within man’s soul. In that it sought to unite these two alchemical realms, the Rosarium sets a style in which the physical process became a mirror for soul development, and the inner content of soul experiencesbecame projected upon outer processes in the laboratory or the natural world. [second paragraph]

Many important biological questions can be addressed by studying in 3D large volumes of intact, cryo fixed hydrated tissues ( >= 10,000 mu m(3)) at high resolution (5-20 nm). This can be achieved using serial FIB milling and block face surface imaging under cryo conditions. Here we demonstrate the unique potential of the cryo-FIB-SEM approach using two extensively studied model systems; sea urchin embryos and the tail fin of zebrafish larvae. We focus in particular on the environment of mineral deposition sites. The cellular organelles, including mitochondria, Golgi, ER, nuclei and nuclear pores are made visible by the image contrast created by differences in surface potential of different biochemical components. Auto segmentation and/or volume rendering of the image stacks and 3D reconstruction of the skeleton and the cellular environment, provides a detailed view of the relative distribution in space of the tissue/cellular components, and thus of their interactions. Simultaneous acquisition of secondary and back-scattered electron images adds additional information. For example, a serial view of the zebrafish tail reveals the presence of electron dense mineral particles inside mitochondrial networks extending more than 20 mu m in depth in the block. Large volume imaging using cryo FIB SEM, as demonstrated here, can contribute significantly to the understanding of the structures and functions of diverse biological tissues. (C) 2016 Elsevier Inc. All rights reserved.

We have developed a high resolution correlative method involving cryo-soft X-ray tomography (cryo-SXT) and stochastic optical reconstruction microscopy (STORM), which provides information in three dimensions on large cellular volumes at 70 nm resolution. Cryo-SXT morphologically identified and localized aggregations of carbon-rich materials. STORM identified specific markers on the desired epitopes, enabling colocalization between the identified objects, in this case cholesterol crystals, and the cellular environment. The samples were studied under ambient and cryogenic conditions without dehydration or heavy metal staining. The early events of cholesterol crystal development were investigated in relation to atherosclerosis, using as model macrophage cell cultures enriched with LDL particles. Atherosclerotic plaques build up in arteries in a slow process involving cholesterol crystal accumulation. Cholesterol crystal deposition is a crucial stage in the pathological cascade. Our results show that cholesterol crystals can be identified and imaged at a very early stage on the cell plasma membrane and in intracellular locations. This technique can in principle be applied to other biological samples where specific molecular identification is required in conjunction with high resolution 3D-imaging.

Both in vivo and ex vivo observations support the hypothesis that bone mineral formation proceeds via disordered precursor phases. The characteristics of the precursor phases are not well defined, but octacalcium phosphate-like, amorphous calcium phosphate-like, and HPO42–-enriched phases were detected. Here we use in vivo Raman spectroscopy and high-resolution wide-angle X-ray diffraction (WAXD) to characterize and map at 2 μm resolution the mineral phases in the rapidly forming tail fin bones of living zebrafish larvae and zebrafish larvae immediately after sacrifice, respectively. Raman spectroscopy shows the presence of an acidic disordered calcium phosphate phase with additional characteristic features of HPO42– at the bone–cell interface. The complexity in the position and shape of the ν1 PO4 peak viewed by in vivo Raman spectroscopy emphasizes the heterogeneity of the mineral during bone formation. WAXD detects an additional isolated peak, appearing alone or together with the characteristic diffraction pattern of carbonated hydroxyapatite. This unidentified phase is located at the interface between the mature bone and the surrounding tissue, similar to the location at which the disordered phase was observed by Raman spectroscopy. The variable peak positions and profiles support the notion that this is an unstable disordered precursor phase, which conceivably crystallized during the X-ray diffraction measurement. Interestingly, this precursor phase is co-aligned with the c-axes of the mature bone crystals and thus is in intimate relation with the surrounding collagen matrix. We conclude that a major disordered precursor mineral phase containing HPO42– is part of the deposition pathway of the rapidly forming tail fin bones of the zebrafish.

The uptake and transport of ions from the environment to the site of bone formation is only partially understood and, for the most part, based on disparate observations in different animals. Here we study different aspects of the biomineralization pathways in one system, the rapidly forming long bones of the chicken embryo. We mainly used cryo-fixation and cryo-electron imaging to preserve the often unstable mineral phases in the tissues. We show the presence of surprisingly large amounts of mineral particles located inside membrane-delineated vesicles in the bone forming tissue between the blood vessels and the forming bone surface. Some of these particles are also located inside mitochondria] networks. The surfaces of the forming bones in the extracellular space contain abundant aggregates of amorphous calcium phosphate particles, but these are not enveloped by vesicle membranes. In the bone resorbing region, osteoclasts also contain many particles in both mitochondria] networks and within vesicles. Some of these particles are present also between cells. These observations, together with the previously reported observation that CaP mineral particles inside membranes are present in blood vessels, leads us to the conclusion that important components of the bone mineralization pathways in rapidly forming chicken bone are dense phase mineral particles bound within membranes. It remains to be determined whether these mineral particles are transported to the site of bone formation in the solid state, fluid state or dissolve and re-precipitate. (C) 2016 Elsevier Inc. All rights reserved.

Light-induced tunable photonic systems are rare in nature, and generally beyond the state-of-the-art in artificial systems. Sapphirinid male copepods produce some of the most spectacular colors in nature. The male coloration, used for communication purposes, is structural and is produced from ordered layers of guanine crystals separated by cytoplasm. It is generally accepted that the colors of the males are related to their location in the epipelagic zone. By combining correlative reflectance and cryoelectron microscopy image analyses, together with optical time lapse recording and transfer matrix modeling, it is shown that male sapphirinids have the remarkable ability to change their reflectance spectrum in response to changes in the light conditions. It is also shown that this color change is achieved by a change in the thickness of the cytoplasm layers that separate the guanine crystals. This change is reversible, and is both intensity and wavelength dependent. This capability provides the male with the ability to efficiently reflect light under certain conditions, while remaining transparent and hence camouflaged under other conditions. These copepods can thus provide inspiration for producing synthetic tunable photonic arrays.

Sea urchin embryos sequester calcium from the sea water. This calcium is deposited in a concentrated form in granule bearing vesicles both in the epithelium and in mesenchymal cells. Here we use in vivo calcein labeling and confocal Raman spectroscopy, as well as cryo-FIB-SEM 3D structural reconstructions, to investigate the processes occurring in the internal cavity of the embryo, the blastocoel. We demonstrate that calcein stained granules are also present in the filopodial network within the blastocoel. Simultaneous fluorescence imaging and Raman spectroscopy show that these granules do contain a calcium mineral. By tracking the movements of these granules, we show that the granules in the epithelium and primary mesenchymal cells barely move, but those in the filopodial network move long distances. We could however not detect any unidirectional movement of the filopodial granules. We also show the presence of mineral containing multivesicular vesicles that also move in the filopodial network. We conclude that the filopodial network is an integral part of the mineral transport process, and possibly also for sequestering calcium and other ions. Although much of the sequestered calcium is deposited in the mineralized skeleton, a significant amount is used for other purposes, and this may be temporarily stored in these membrane-delineated intracellular deposits. (C) 2015 Elsevier Inc. All rights reserved.

Living organisms display a spectrum of wondrous colors, which can be produced by pigmentation, structural coloration, or a combination of the two. A relatively well-studied system, which produces colors via an array of alternating anhydrous guanine crystals and cytoplasm, is responsible for the metallic luster of many fish. The structure of biogenic anhydrous guanine was so far believed to be the same as that of the synthetic one, a monoclinic polymorph (denoted as alpha). Here we re-examine the structure of biogenic guanine, using detailed experimental X-ray and electron diffraction data, exposing troublesome inconsistencies, namely, a "guanigma". To address this, we sought alternative candidate polymorphs using symmetry and packing considerations and then utilized first-principles calculations to determine whether the selected candidates could be energetically stable. We identified theoretically a different monoclinic polymorph (denoted as beta), were able to synthesize it, and confirmed using X-ray diffraction that it is this polymorph that occurs in biogenic samples. However, the electron diffraction data were still not consistent with this polymorph but rather with a theoretically generated orthorhombic polymorph (denoted as gamma). This apparent inconsistency was resolved by showing how the electron diffraction pattern could be affected by crystal structural faults composed of offset molecular layers.

Males of sapphirinid copepods use regularly alternating layers of hexagonal-shaped guanine crystals and cytoplasm to produce spectacular structural colors. In order to understand the mechanism by which the different colors are produced, we measured the reflectance of live individuals and then characterized the organization of the crystals and the cytoplasm layers in the same individuals using cryo-SEM. On the basis of these measurements, we calculated the expected reflectance spectra and found that they are strikingly similar to the measured ones. We show that variations in the cytoplasm layer thickness are mainly responsible for the different reflected colors and also that the copepod color strongly depends on the angular orientation relative to the incident light, which can account for its appearance and disappearance during spiral swimming in the natural habitat.

Calcium carbonate is a common constituent of many natural materials, such as shells and skeletons of marine animals. While it is well-documented that additives (organic and inorganic) modulate the crystallization of amorphous calcium carbonate (ACC), the effects of the intrinsic physicochemical characteristics of ACC, such as particle size, shape, and water content on the transformation to crystalline polymorphs, are still poorly understood. Here, we investigate the effect of particle size by preparing ACC nanopartides with an average size ranging from similar to 66 to similar to 196 nm using a high-resolution titration setup. Our results show that the particle size determined the polymorph selection in solution; an increasing proportion of vaterite to calcite was observed with decreasing particle size. The polymorph selection was ascribed to a higher apparent solubility of ACC with decreasing particle size, a parameter from which we could determine the surface energy of ACC to be similar to 0.33 J/m(2). Upon heating, particle size showed the opposite effect, as smaller particles favored a higher crystallization temperature from ACC into (only) calcite. When the particle size was large enough, crystallization occurred concomitantly with the removal of bulk water at lower temperatures, where the smallest particles transformed at similar to 310 degrees C, only after losing the final (surface) water. Our results highlight the importance of particle size as well as the crystallization conditions on the stability and transformation mechanisms of ACC.

Atherosclerosis is the major precursor of cardiovascular disease. The formation of cholesterol crystals in atherosclerotic plaques is associated with the onset of acute pathology. The cholesterol crystals induce physical injury in the plaque core, promoting cell apoptosis and triggering an increased inflammatory response. Herein we address the question of how cholesterol crystal formation occurs in atherosclerosis. We demonstrate that three-dimensional (3D) cholesterol crystals can undergo directed nucleation from bilayer membranes containing two-dimensional (2D) cholesterol crystalline domains. We studied crystal formation on supported lipid bilayers loaded with exogenous cholesterol and labeled using a monoclonal antibody that specifically recognizes ordered cholesterol arrays. Our findings show that 3D crystals are formed exclusively on the bilayer regions where there are segregated 2D cholesterol crystalline domains and that they form on the domains. This study has potentially significant implications for our understanding of the crucial step in the mechanism by which atherosclerotic lesions form.

Organisms tune the metastability of amorphous calcium carbonates (ACC), often by incorporation of additives such as phosphate ions and water molecules, to serve diverse functions, such as modulating the availability of calcium reserves or constructing complex skeletal scaffolds. Although the effect of additive distribution on ACC was noted for several biogenic and synthetic systems, the molecular mechanisms by which additives govern ACC stability are not well understood. By precipitating ACC in the presence of different PO43 concentrations and regulating the initial water content, we identify conditions yielding either kinetically locked or spontaneously transforming coprecipitates. Solid state NMR, supported by FTIR, XRD, and electron microscopy, define the interactions of phosphate and water within the initial amorphous matrix, showing that initially the coprecipitates are homogeneous molecular dispersions of structural water and phosphate in ACC, and a small fraction of P-rich phases. Monitoring the transformations of the homogeneous phase shows that PO43 and waters are extracted first, and they phase separate, leading to solidsolid transformation of ACC to calcite; small part of ACC forms vaterite that subsequently converts to calcite. The simultaneous waterPO(4)(3) extraction is the key for the subsequent water-mediated accumulation and crystallization of hydroxyapatite (HAp) and carbonated hydroxyapatite. The thermodynamic driving force for the transformations is calcite crystallization, yet it is gated by specific combinations of waterphosphate levels in the initial amorphous coprecipitates. The molecular details of the spontaneously transforming ACC and of the stabilized ACC modulated by phosphate and water at ambient conditions, provide insight into biogenic and biomimetic pathways.

Many biogenic minerals are composed of aggregated particles at the nanoscale. These minerals usually form through the transformation of amorphous precursors into single crystals inside a privileged space controlled by the organism. Here, in vitro experiments aimed at understanding the factors responsible for producing such single crystals with aggregated particle texture are presented. Crystallization is achieved by a two-step reaction in which amorphous calcium carbonate (ACC) is first precipitated and then transformed into calcite in small volumes of water and in the presence of additives. The additives used are gel-forming molecules, phosphate ions, and the organic extract from sea urchin embryonic spicules - all are present in various biogenic crystals that grow via the transformation of ACC. Remarkably, this procedure yields faceted single-crystals of calcite that maintain the nanoparticle texture. The crystals grow predominantly by the accretion of ACC nanoparticles, which subsequently crystallize. Gels and phosphate ions stabilize ACC via a different mechanism than sea urchin spicule macromolecules. It is concluded that the unique nanoparticle texture of biogenic minerals results from formation pathways that may differ from one another, but given the appropriate precursor and micro-environment, share a common particle accretion mechanism.

X-ray absorption near-edge structure (XANES) spectroscopy and spectromicroscopy have been extensively used to characterize biominerals. Using either Ca or C spectra, unique information has been obtained regarding amorphous biominerals and nanocrystal orientations. Building on these results, we demonstrate that recording XANES spectra of calcium carbonate at the oxygen K-edge enables polarization-dependent imaging contrast (PIC) mapping with unprecedented contrast, signal-to-noise ratio, and magnification. O and Ca spectra are presented for six calcium carbonate minerals: aragonite, calcite, vaterite, monohydrocalcite, and both hydrated and anhydrous amorphous calcium carbonate. The crystalline minerals reveal excellent agreement of the extent and direction of polarization dependences in simulated and experimental XANES spectra due to X-ray linear dichroism. This effect is particularly strong for aragonite, calcite, and vaterite. In natural biominerals, oxygen PIC-mapping generated high-magnification maps of unprecedented clarity from nacre and prismatic structures and their interface in Mytilus californianus shells. These maps revealed blocky aragonite crystals at the nacre-prismatic boundary and the narrowest calcite needle-prisms. In the tunic spicules of Herdmania momus, O PIC-mapping revealed the size and arrangement of some of the largest vaterite single crystals known. O spectroscopy therefore enables the simultaneous measurement of chemical and orientational information in CaCO3 biominerals and is thus a powerful means for analyzing these and other complex materials. As described here, PIC-mapping and spectroscopy at the O K-edge are methods for gathering valuable data that can be carried out using spectromicroscopy beamlines at most synchrotrons without the expense of additional equipment.

Brushite, CaHPO4 center dot 2H(2)O, is a crystalline hydrated acidic form of calcium phosphate that occurs in both physiological and pathological biomineralization processes. Additionally, it is biocompatible in humans. Several groups have investigated the experimental Fourier transform infrared vibrational spectrum of brushite. These investigations have led to a long-standing debate concerning the correct assignment for a few of the observed frequencies, particularly, the water stretching and libration modes. Here, we perform a comprehensive first principles theoretical investigation of the vibrational spectrum of brushite with calculations based on dispersion-corrected density functional theory. We obtain both the vibrational frequencies, using the frozen phonon approach, and their corresponding peak intensities, based on the Born effective charge tensor. This allows for an unambiguous assignment of all vibrations, including water vibration and libration modes.

Farber disease is an inherited metabolic disorder caused by mutations in the acid ceramidase gene, which leads to ceramide accumulation in lysosomes. Farber disease patients display a wide variety of symptoms with most patients eventually displaying signs of nervous system dysfunction. We now present a novel tool that could potentially be used to distinguish between the milder and more severe forms of the disease, namely, an antibody that recognizes a mixed monolayer or bilayer of cholesterol: C16-ceramide, but does not recognize either ceramide or cholesterol by themselves. This antibody has previously been used to detect cholesterol: C16-ceramide domains in a variety of cultured cells. We demonstrate that levels of cholesterol: C16-ceramide domains are significantly elevated in fibroblasts from types 4 and 7 Farber disease patients, and that levels of the domains can be modulated by either reducing ceramide or cholesterol levels. Moreover, these domains are located in membranes of the endomembrane system, and also in two unexpected locations, namely, the mitochondria and the plasma membrane. This study suggests that the ceramide that accumulates in severe forms of Farber disease cells is sequestered to distinct membrane subdomains, which may explain some of the cellular pathology observed in this devastating lysosomal storage disease.

Bone degradation by osteoclasts depends on the formation of a sealing zone, composed of an interlinked network of podosomes, which delimits the degradation lacuna into which osteoclasts secrete acid and proteolytic enzymes. For resorption to occur, the sealing zone must be coherent and stable for extended periods of time. Using titanium roughness gradients ranging from 1 to 4.5 mu m R-a as substrates for osteoclast adhesion, we show that microtopographic obstacles of a length scale well beyond the range of the 'footprint' of an individual podosome can slow down sealing-zone expansion. A clear inverse correlation was found between ring stability, structural integrity and sealing-zone translocation rate. Direct live-cell microscopy indicated that the expansion of the sealing zone is locally arrested by steep, three-dimensional 'ridge-like barriers', running parallel to its perimeter. It was, however, also evident that the sealing zone can bypass such obstacles, if pulled by neighbouring regions, extending through flanking, obstacle-free areas. We propose that sealing-zone dynamics, while being locally regulated by surface roughness, are globally integrated via the associated actin cytoskeleton. The effect of substrate roughness on osteoclast behaviour is significant in relation to osteoclast function under physiological and pathological conditions, and may constitute an important consideration in the design of advanced bone replacements.

Podosomes, which are formed by different monocyte derivatives, are small adhesion structures whose coordinated dynamics and cytoskeletal reorganization drive their motile and invasive features. Using live-cell microscopy, we explored the temporal molecular steps of the de novo assembly and disassembly of podosomes in cultured osteoclasts. We demonstrate here that the earliest visible step in podosome assembly is the local accumulation of the plaque protein paxillin, along with cortactin, which stabilizes actin networks, followed by robust polymerization of actin filaments and their association with alpha-actinin. Only then is a local increase in integrin beta 3 levels apparent in the podosome ring domain. Thus, local actin polymerization in cortactin-and paxillin-rich locations nucleates podosome assembly before the local accumulation of beta 3 integrin. We further show that actin polymerization is also important for the recruitment and maintenance of plaque proteins in the mature podosome ring domain. Our model implies that core bundle dynamics play a central role in regulating podosome stability.