Bio-Inspired Crystalline Core-Shell Guanine Spherulites.

Spherical particles with diameters within the wavelength of visible light, known as spherulites, manipulate light uniquely due to their spatial organization and their structural birefringence. Most of the known crystalline spherulites are branched, and composed of metals, alloys, and semi-crystalline polymers. Recently, a different spherulite architecture is discovered in the vision systems of decapod crustaceans - core-shell spherulites composed of highly birefringent ( Δ n ≈ 30 % $\Delta n \approx \ 30\%$ ) organic single-crystal platelets, with exceptional optical properties. These metastructures, which efficiently scatter light even in dense aqueous environments, have no synthetic equivalence and serve as a natural proof-of-concept as well as synthetic inspiration for thin scattering media. Here, the synthesis of core-shell spherulites composed of guanine crystal platelets (( Δ n ≈ 25 % $\Delta n \approx 25\%$ ) is presented in a two-step emulsification process in which a water/oil/water emulsion and induced pH changes are used to promote interfacial crystallization. Carboxylic acids neutralize the dissolved guanine salts to form spherulites composed of single, radially stacked, ß-guanine platelets, which are oriented tangentially to the spherulite surface. Using Mie theory calculations and forward scattering measurements from single spherulites, it is found that due to the single-crystal properties and orientation, the synthetic spherulites possess a high tangential refractive index, similarly to biogenic particles.

Elemental compositions of sea urchin larval cell vesicles evaluated by cryo-STEM-EDS and cryo-SEM-EDS.

During spicule formation in sea urchin larvae, calcium ions translocate within the primary mesenchymal cells (PMCs) from endocytosed seawater vacuoles to various organelles and vesicles where they accumulate, and subsequently precipitate. During this process, calcium ions are concentrated by more than three orders of magnitude, while other abundant ions (Na, Mg) must be removed. To obtain information about the overall ion composition in the vesicles, we used quantitative cryo-SEM-EDS and cryo-STEM-EDS analyzes. For cryo-STEM-EDS, thin (500 nm) frozen hydrated lamellae of PMCs were fabricated using cryo-focused ion beam-SEM. The lamellae were then loaded into a cryo-TEM, imaged and the ion composition of electron dense bodies was measured. Analyzes performed on 18 Ca-rich particles/particle clusters from 6 cells contained Ca, Na, Mg, S and P in different ratios. Surprisingly, all the Ca-rich particles contained P in amounts up to almost 1:1 of Ca. These cryo-STEM-EDS results were qualitatively confirmed by cryo-SEM-EDS analyzes of 310 vesicles, performed on high pressure frozen and cryo-planed samples. We discuss the advantages and limitations of the two techniques, and their potential applicability, especially to study ion transport pathways and ion trafficking in cells involved in mineralization. STATEMENT OF SIGNIFICANCE: The 'inorganic side of life', encompassing ion trafficking and ion storage in soft tissues of organisms, is a generally overlooked problem. Addressing such a problem becomes possible through the application of innovative techniques, performed in cryogenic conditions, which preserve the tissues in quasi-physiological state. We developed here a set of analytical tools, cryo-SEM-EDS, and cryo-STEM-EDS, which allow reconstructing the ion composition inside vesicles in sea urchin larval cells, on their way to deposit mineral in the skeletons. The techniques are complex, and we evaluate here the advantages and disadvantages of each technique. The methodologies that we are developing here can be applied to other cells and other pathways as well, eventually leading to quantitative elemental analyzes of tissues under cryogenic conditions.

Examining atherosclerotic lesions in three dimensions at the nanometer scale with cryo-FIB-SEM.

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.

Polymorphism, Structure, and Nucleation of Cholesterol·H2O at Aqueous Interfaces and in Pathological Media: Revisited from a Computational Perspective.

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.

Structural organization of xanthine crystals in the median ocellus of a member of the ancestral insect group Archaeognatha.

Biogenic purine crystals function in vision as mirrors, multilayer reflectors and light scatterers. We investigated a light sensory organ in a primarily wingless insect, the jumping bristletail Lepismachilis rozsypali (Archaeognatha), an ancestral group. The visual system of this animal comprises two compound eyes, two lateral ocelli, and a median ocellus, which is located on the front of the head, pointing downwards to the ground surface. We determined that the median ocellus contains crystals of xanthine, and we obtained insights into their function. To date, xanthine biocrystals have only been found in the Archaeognatha. We performed a structural analysis, using reflection light microscopy, cryo-FIB-SEM, microCT and cryo-SEM. The xanthine crystals cover the bottom of a bowl-shaped volume in the median ocellus, in analogy to a tapetum, and reflect photons to light-sensitive receptors that are spread in the volume without apparent order or preferential orientation. We infer that the median ocellus operates as an irregular multifocal reflector, which is not capable of forming images. A possible function of this organ is to improve photon capture, and by so doing assess distances from the ground surface when jumping by determining changes in the intensity and contrast of the incident light.

Ion Pathways in Biomineralization: Perspectives on Uptake, Transport, and Deposition of Calcium, Carbonate, and Phosphate.

Minerals are formed by organisms in all of the kingdoms of life. Mineral formation pathways all involve uptake of ions from the environment, transport of ions by cells, sometimes temporary storage, and ultimately deposition in or outside of the cells. Even though the details of how all this is achieved vary enormously, all pathways need to respect both the chemical limitations of ion manipulation, as well as the many "housekeeping" roles of ions in cell functioning. Here we provide a chemical perspective on the biological pathways of biomineralization. Our approach is to compare and contrast the ion pathways involving calcium, phosphate, and carbonate in three very different organisms: the enormously abundant unicellular marine coccolithophores, the well investigated sea urchin larval model for single crystal formation, and the complex pathways used by vertebrates to form their bones. The comparison highlights both common and unique processes. Significantly, phosphate is involved in regulating calcium carbonate deposition and carbonate is involved in regulating calcium phosphate deposition. One often overlooked commonality is that, from uptake to deposition, the solutions involved are usually supersaturated. This therefore requires not only avoiding mineral deposition where it is not needed but also exploiting this saturated state to produce unstable mineral precursors that can be conveniently stored, redissolved, and manipulated into diverse shapes and upon deposition transformed into more ordered and hence often functional final deposits.

Characterization of the growth plate-bone interphase region using cryo-FIB SEM 3D volume imaging.

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.

Measuring the optical properties of nanoscale biogenic spherulites.

Recent studies of optical reflectors as part of the vision apparatus in the eyes of decapod crustaceans revealed assemblies of nanoscale spherulites - spherical core-shell nanoparticles with radial birefringence. Simulations performed on the system highlighted the advantages of optical anisotropy in enhancing the functionality of these structures. So far, calculations of the nanoparticle optical properties have relied on refractive indices obtained using ab-initio calculations. Here we describe a direct measurement of the tangential refractive index of the spherulites, which corresponds to the in-plane refractive index of crystalline isoxanthopterin nanoplatelets. We utilize measurements of scattering spectra of individual spherulites and determine the refractive index by analyzing the spectral signatures of scattering resonances. Our measurements yield a median tangential refractive index of 1.88, which is in reasonable agreement with theoretical predictions. Furthermore, our results indicate that the optical properties of small spherulite assemblies are largely determined by the tangential index.

Fluorescent Silica Nanoparticles to Label Metastatic Tumor Cells in Mineralized Bone Microenvironments.

During breast cancer bone metastasis, tumor cells interact with bone microenvironment components including inorganic minerals. Bone mineralization is a dynamic process and varies spatiotemporally as a function of cancer-promoting conditions such as age and diet. The functional relationship between skeletal dissemination of tumor cells and bone mineralization, however, is unclear. Standard histological analysis of bone metastasis frequently relies on prior demineralization of bone, while methods that maintain mineral are often harsh and damage fluorophores commonly used to label tumor cells. Here, fluorescent silica nanoparticles (SNPs) are introduced as a robust and versatile labeling strategy to analyze tumor cells within mineralized bone. SNP uptake and labeling efficiency of MDA-MB-231 breast cancer cells is characterized with cryo-scanning electron microscopy and different tissue processing methods. Using a 3D in vitro model of marrow-containing, mineralized bone as well as an in vivo model of bone metastasis, SNPs are demonstrated to allow visualization of labeled tumor cells in mineralized bone using various imaging modalities including widefield, confocal, and light sheet microscopy. This work suggests that SNPs are valuable tools to analyze tumor cells within mineralized bone using a broad range of bone processing and imaging techniques with the potential to increase the understanding of bone metastasis.

Cellular pathways of calcium transport and concentration toward mineral formation in sea urchin larvae.

Sea urchin larvae have an endoskeleton consisting of two calcitic spicules. The primary mesenchyme cells (PMCs) are the cells that are responsible for spicule formation. PMCs endocytose sea water from the larval internal body cavity into a network of vacuoles and vesicles, where calcium ions are concentrated until they precipitate in the form of amorphous calcium carbonate (ACC). The mineral is subsequently transferred to the syncytium, where the spicule forms. Using cryo-soft X-ray microscopy we imaged intracellular calcium-containing particles in the PMCs and acquired Ca-L2,3 X-ray absorption near-edge spectra of these Ca-rich particles. Using the prepeak/main peak (L2'/ L2) intensity ratio, which reflects the atomic order in the first Ca coordination shell, we determined the state of the calcium ions in each particle. The concentration of Ca in each of the particles was also determined by the integrated area in the main Ca absorption peak. We observed about 700 Ca-rich particles with order parameters, L2'/ L2, ranging from solution to hydrated and anhydrous ACC, and with concentrations ranging between 1 and 15 M. We conclude that in each cell the calcium ions exist in a continuum of states. This implies that most, but not all, water is expelled from the particles. This cellular process of calcium concentration may represent a widespread pathway in mineralizing organisms.

Characterization and possible function of an enigmatic reflector in the eye of the shrimp Litopenaeus vannamei.

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.

A highly reflective biogenic photonic material from core-shell birefringent nanoparticles.

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.

Mineralization pathways in the active murine epiphyseal growth plate.

Endochondral ossification in the growth plate of long bones involves cartilage mineralization, bone formation and the budding vasculature. Many of these processes take place in a complex and dynamic zone, the provisional ossification zone, of the growth plate. Here we investigate aspects of mineralization in 2D and 3D in the provisional ossification zone at different length scales using samples preserved under cryogenic or fully hydrated conditions. We use confocal light microscopy, cryo-SEM and micro-CT in the phase contrast mode. We show in 9 week old BALB/c mice the presence of vesicles containing mineral particles in the blood serum, as well as mineral particles without membranes integrated with the blood vessel walls. We also observe labeled mineral particles within cells associated with bone formation, but not in the hypertrophic cartilage cells that are involved with cartilage mineralization. High resolution micro-CT images of fresh hydrated tibiae, show that there are open continuous pathways between the blood vessel extremities and the hypertrophic chondrocyte zone. As the blood vessel extremities, the mineralizing cartilage and the forming bone are all closely associated within this narrow zone, we raise the possibility that in addition to ion transport, mineral necessary for both cartilage and bone formation is also transported through the vasculature.

Biomineralization pathways in calcifying dinoflagellates: Uptake, storage in MgCaP-rich bodies and formation of the shell.

Little is known about shell formation of calcareous dinoflagellates, despite the fact that they are one of the major calcifying organisms of the phytoplankton. Here, calcitic cyst formation in two representative members of calcareous dinoflagellates is investigated using cryo-electron microscopy (cryo-SEM and cryo-FIB-SEM) in combination with micro-Raman and infrared spectroscopy. Only calcein-AM and not calcein enters these cells, indicating active uptake of calcium and other divalent cations. Multifunctional vacuoles containing crystalline inclusions are observed, and the crystals are identified as anhydrous guanine in the ß-form. The same vacuolar enclosures contain dense magnesium-, calcium-, and phosphorous-rich mineral bodies. These bodies are presumably secreted into the outer matrix where calcite forms. Calcite formation occurs via multiple independent nucleation events, and the different crystals grow with preferred orientation into a dense reticular network that forms the mature calcitic shell. We suggest a biomineralization pathway for calcareous dinoflagellates that includes (1) active uptake of calcium through the membranes, (2) deposition of Mg2+- and Ca2+-ions inside disordered MgCaP-rich mineral bodies, (3) secretion of these bodies to the inter-membrane space, and (4) Formation and growth of calcite into a dense reticulate network. This study provides new insights into calcium uptake, storage and transport in calcifying dinoflagellates. STATEMENT OF SIGNIFICANCE: Little is known about the shell formation of calcareous dinoflagellates, despite the fact that they are one of the major calcifying organisms of the phytoplankton. We used state-of-the-art cryo-electron microscopy (cryo-SEM and cryo-FIB-SEM) in combination with micro-Raman spectroscopy to provide new insights into mineral formation in calcifying dinoflagellates. To date, intracellular crystalline calcite was assumed to be involved in calcite shell formation. Surprisingly, we identify these crystalline inclusions as anhydrous guanine suggesting that they are not involved in biomineralization. Instead, a key finding is that MgCaP-rich bodies are probably secreted into the outer matrix where the calcite shell is formed. We suggest that these bodies are an essential part of Ca-uptake, -storage and -transport and propose a new biomineralization model.

Guanine and 7,8-Dihydroxanthopterin Reflecting Crystals in the Zander Fish Eye: Crystal Locations, Compositions, and Structures.

The eyes of many fish contain a reflecting layer of organic crystals partially surrounding the photoreceptors of the retina, which are commonly believed to be composed of guanine. Here we study an unusual fish eye from Stizostedion lucioperca that contains two layers of organic crystals. The crystals in the outer layer are thin plates, whereas the crystals in the inner tapetum layer are block-shaped. We show that the outer layer indeed contains guanine crystals. Analyses of solutions of crystals from the inner layer indicated that the block-shaped crystals are composed of xanthopterin. A model of the structure of the block-shaped crystals was produced using symmetry arguments based on electron diffraction data followed by dispersion-augmented DFT calculations. The resulting crystal structure of xanthopterin included, however, a problematic repulsive interaction between C═O and N of two adjacent molecules. Knowing that dissolved 7,8-dihydroxanthopterin can oxidize to xanthopterin, we replaced xanthopterin with 7,8-dihydroxanthopterin in the model. An excellent fit was obtained with the powder X-ray diffraction pattern of the biogenic crystals. We then analyzed the biogenic block-shaped crystals in their solid state, using MALDI-TOF and Raman spectroscopy. All three methods unequivocally prove that the block-shaped crystals in the eye of S. lucioperca are crystals of 7,8-dihydroxanthopterin. On the basis of the eye anatomy, we deduce that the guanine crystals form a reflective layer producing the silvery color present on part of the eye surface, whereas the block-shaped crystals backscatter light into the retina in order to increase the light sensitivity of the eye.

Anhydrous ß-guanine crystals in a marine dinoflagellate: Structure and suggested function.

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 ß-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.

Intercellular pathways from the vasculature to the forming bone in the zebrafish larval caudal fin: Possible role in bone formation.

The pathway of ion supply from the source to the site of bone deposition in vertebrates is thought to involve transport through the vasculature, followed by ion concentration in osteoblasts. The cells deposit a precursor mineral phase in vesicles, which are then exocytosed into the extracellular matrix. We observed that the entire skeleton of zebrafish larvae, is labelled within minutes after injection of calcein or FITC-dextran into the blood. This raised the possibility that there is an additional pathway of solute transport that can account for the rapid labelling. We used cryo-FIB-SEM serial block face imaging to reconstruct at high resolution the 3D ultrastructure of the caudal tail of the zebrafish larva. This reconstruction clearly shows that there is a continuous intercellular pathway from the artery to the forming bone, and from the forming bone to the vein. Fluorescence light microscopy shows that calcein and FITC-dextran form a reticulate network pattern in this tissue, which we attribute to the dye being present in the intercellular space. We conclude that this intercellular continuous space may be a supply route for ions, mineral and other solute or particulate material to the fast forming bone.

A 3D study of the relationship between leaf vein structure and mechanical function.

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 leaf's 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 SIGNIFICANCE: Leaves 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.

The Effect of the Phospholipid Bilayer Environment on Cholesterol Crystal Polymorphism.

Cholesterol crystallization from mixtures of unesterified cholesterol with phospholipids and cholesterol esters is believed to be a key event in atherosclerosis progression. Not much is understood, however, about the influence of the lipid environment on cholesterol crystallization. Here we study cholesterol monohydrate crystal formation from mixed bilayers with palmitoyl-oleoyl-phosphatidylcholine (POPC), dipalmitoyl-phosphatidylcholine (DPPC) and sphingomyelin. We show that disordered phospholipids and sphingomyelin stabilize the formation of crystal plates of the triclinic cholesterol monohydrate polymorph, whereas saturated glycerolipids stabilize helical and tubular crystals of the metastable monoclinic polymorph. We followed the subsequent transformation of these helical crystals into the stable triclinic plates. Discovering the relations between membrane lipid composition and cholesterol crystal polymorphism may provide important clues to the understanding of cholesterol crystal formation in atherosclerosis.

The Effect of the Phospholipid Bilayer Environment on Cholesterol Crystal Polymorphism.

Invited for this month's cover are the group of Prof. Lia Addadi at the Weizmann Institute of Science, Israel and collaborators at the Università Degli Studi di Milano, Italy, and the ALBA Synchrotron Light Source, Spain. The front cover shows how cholesterol crystals form in macrophage cells and in lipid bilayers of different compositions. Cholesterol monohydrate stable triclinic crystals form in vitro as rhomb-shaped plates, whereas the monoclinic crystals fold into tubular or helical shapes. Read the full text of the article at 10.1002/cplu.201800632.