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.

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.

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.

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.

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.

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.

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.

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.

Optically functional isoxanthopterin crystals in the mirrored eyes of decapod crustaceans.

The eyes of some aquatic animals form images through reflective optics. Shrimp, lobsters, crayfish, and prawns possess reflecting superposition compound eyes, composed of thousands of square-faceted eye units (ommatidia). Mirrors in the upper part of the eye (the distal mirror) reflect light collected from many ommatidia onto the photosensitive elements of the retina, the rhabdoms. A second reflector, the tapetum, underlying the retina, back-scatters dispersed light onto the rhabdoms. Using microCT and cryo-SEM imaging accompanied by in situ micro-X-ray diffraction and micro-Raman spectroscopy, we investigated the hierarchical organization and materials properties of the reflective systems at high resolution and under close-to-physiological conditions. We show that the distal mirror consists of three or four layers of plate-like nanocrystals. The tapetum is a diffuse reflector composed of hollow nanoparticles constructed from concentric lamellae of crystals. Isoxanthopterin, a pteridine analog of guanine, forms both the reflectors in the distal mirror and in the tapetum. The crystal structure of isoxanthopterin was determined from crystal-structure prediction calculations and verified by comparison with experimental X-ray diffraction. The extended hydrogen-bonded layers of the molecules result in an extremely high calculated refractive index in the H-bonded plane, n = 1.96, which makes isoxanthopterin crystals an ideal reflecting material. The crystal structure of isoxanthopterin, together with a detailed knowledge of the reflector superstructures, provide a rationalization of the reflective optics of the crustacean eye.

Two polymorphic cholesterol monohydrate crystal structures form in macrophage culture models of atherosclerosis.

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 rod-like 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.

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.

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.

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.

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.

Mineral Deposits in Ficus Leaves: Morphologies and Locations in Relation to Function.

Ficus trees are adapted to diverse environments and have some of the highest rates of photosynthesis among trees. Ficus leaves can deposit one or more of the three major mineral types found in leaves: amorphous calcium carbonate cystoliths, calcium oxalates, and silica phytoliths. In order to better understand the functions of these minerals and the control that the leaf exerts over mineral deposition, we investigated leaves from 10 Ficus species from vastly different environments (Rehovot, Israel; Bologna, Italy; Issa Valley, Tanzania; and Ngogo, Uganda). We identified the mineral locations in the soft tissues, the relative distributions of the minerals, and mineral volume contents using microcomputed tomography. Each Ficus species is characterized by a unique 3D mineral distribution that is preserved in different environments. The mineral distribution patterns are generally different on the adaxial and abaxial sides of the leaf. All species examined have abundant calcium oxalate deposits around the veins. We used micromodulated fluorimetry to examine the effect of cystoliths on photosynthetic efficiency in two species having cystoliths abaxially and adaxially (Ficusmicrocarpa) or only abaxially (Ficuscarica). In F. microcarpa, both adaxial and abaxial cystoliths efficiently contributed to light redistribution inside the leaf and, hence, increased photosynthetic efficiency, whereas in F. carica, the abaxial cystoliths did not increase photosynthetic efficiency.

Macrophages Shed Excess Cholesterol in Unique Extracellular Structures Containing Cholesterol Microdomains.

OBJECTIVE: Cells use various mechanisms to maintain cellular cholesterol homeostasis including efflux of cholesterol from the cellular plasma membrane to cholesterol acceptors such as HDLs (high-density lipoproteins). Little is known about the transfer of cholesterol from cells into the extracellular matrix. Using a unique monoclonal antibody that detects ordered cholesterol arrays (ie, cholesterol micro[or nano]-domains), we previously identified that particles containing these cholesterol domains accumulate in the extracellular matrix during cholesterol enrichment of human monocyte-derived macrophages and are found in atherosclerotic lesions. In this study, we further investigate these deposited particles containing cholesterol microdomains and discover their unexpected morphology. APPROACH AND RESULTS: Although appearing spherical at the resolution of the conventional fluorescence microscope, super-resolution immunofluorescence and atomic force microscopy of in situ cholesterol microdomains, and immunoelectron microscopy of isolated cholesterol microdomains revealed that the microdomains are not vesicles or 3-dimensional crystals but rather appear as branching irregularly shaped deposits of varying size. These cholesterol microdomain-containing deposits are shed from the plasma membrane into the extracellular matrix. CONCLUSIONS: To date, research on cellular excretion of excess cholesterol has demonstrated cellular cholesterol efflux in the form of membranous vesicles and discoidal HDL particles released into the fluid-phase medium. Shedding of plasma membrane cholesterol microdomains provides an additional mechanism for cells such as macrophages to maintain plasma membrane cholesterol homeostasis. Furthermore, recognition that macrophages shed cholesterol microdomains into the extracellular matrix is important to our understanding of extracellular buildup of cholesterol in atherosclerosis.

Calcium transport into the cells of the sea urchin larva in relation to spicule formation.

We investigated the manner in which the sea urchin larva takes up calcium from its body cavity into the primary mesenchymal cells (PMCs) that are responsible for spicule formation. We used the membrane-impermeable fluorescent dye calcein and alexa-dextran, with or without a calcium channel inhibitor, and imaged the larvae in vivo with selective-plane illumination microscopy. Both fluorescent molecules are taken up from the body cavity into the PMCs and ectoderm cells, where the two labels are predominantly colocalized in particles, whereas the calcium-binding calcein label is mainly excluded from the endoderm and is concentrated in the spicules. The presence of vesicles and vacuoles inside the PMCs that have openings through the plasma membrane directly to the body cavity was documented using high-resolution cryo-focused ion beam-SEM serial imaging. Some of the vesicles and vacuoles are interconnected to form large networks. We suggest that these vacuolar networks are involved in direct sea water uptake. We conclude that the calcium pathway from the body cavity into cells involves nonspecific endocytosis of sea water with its calcium.

ABCA1 (ATP-Binding Cassette Transporter A1) Mediates ApoA-I (Apolipoprotein A-I) and ApoA-I Mimetic Peptide Mobilization of Extracellular Cholesterol Microdomains Deposited by Macrophages.

OBJECTIVE: We examined the function of ABCA1 (ATP-binding cassette transporter A1) in ApoA-I (apolipoprotein A-I) mobilization of cholesterol microdomains deposited into the extracellular matrix by cholesterol-enriched macrophages. We have also determined whether an ApoA-I mimetic peptide without and with complexing to sphingomyelin can mobilize macrophage-deposited cholesterol microdomains. APPROACH AND RESULTS: Extracellular cholesterol microdomains deposited by cholesterol-enriched macrophages were detected with a monoclonal antibody, 58B1. ApoA-I and an ApoA-I mimetic peptide 5A mobilized cholesterol microdomains deposited by ABCA1+/+ macrophages but not by ABCA1-/- macrophages. In contrast, ApoA-I mimetic peptide 5A complexed with sphingomyelin could mobilize cholesterol microdomains deposited by ABCA1-/- macrophages. CONCLUSIONS: Our findings show that a unique pool of extracellular cholesterol microdomains deposited by macrophages can be mobilized by both ApoA-I and an ApoA-I mimetic peptide but that mobilization depends on macrophage ABCA1. It is known that ABCA1 complexes ApoA-I and ApoA-I mimetic peptide with phospholipid, a cholesterol-solubilizing agent, explaining the requirement for ABCA1 in extracellular cholesterol microdomain mobilization. Importantly, ApoA-I mimetic peptide already complexed with phospholipid can mobilize macrophage-deposited extracellular cholesterol microdomains even in the absence of ABCA1.

Initial stages of calcium uptake and mineral deposition in sea urchin embryos.

Sea urchin larvae have an endoskeleton consisting of two calcitic spicules. We reconstructed various stages of the formation pathway of calcium carbonate from calcium ions in sea water to mineral deposition and integration into the forming spicules. Monitoring calcium uptake with the fluorescent dye calcein shows that calcium ions first penetrate the embryo and later are deposited intracellularly. Surprisingly, calcium carbonate deposits are distributed widely all over the embryo, including in the primary mesenchyme cells and in the surface epithelial cells. Using cryo-SEM, we show that the intracellular calcium carbonate deposits are contained in vesicles of diameter 0.5-1.5 µm. Using the newly developed airSEM, which allows direct correlation between fluorescence and energy dispersive spectroscopy, we confirmed the presence of solid calcium carbonate in the vesicles. This mineral phase appears as aggregates of 20-30-nm nanospheres, consistent with amorphous calcium carbonate. The aggregates finally are introduced into the spicule compartment, where they integrate into the growing spicule.

Biologically Controlled Morphology and Twinning in Guanine Crystals.

Guanine crystals are widely used in nature as components of multilayer reflectors. Guanine-based reflective systems found in the copepod cuticle and in the mirror of the scallop eye are unique in that the multilayered reflectors are tiled to form a contiguous packed array. In the copepod cuticle, hexagonal crystals are closely packed to produce brilliant colors. In the scallop eye, square crystals are tiled to obtain an image-forming reflecting mirror. The tiles are about 1 µm in size and 70 nm thick. According to analysis of their electron diffraction patterns, the hexagon and square tiles are not single crystals. Rather, each tile type is a composite of what appears to be three crystalline domains differently oriented and stacked onto one another, achieved through a twice-repeated twinning about their ⟨011⟩ and ⟨021⟩ crystal axes, respectively. By these means, the monoclinic guanine crystal mimics higher symmetry hexagonal and tetragonal structures to achieve unique morphologies.