High-resolution electron microscopic investigation of crystalline cholesterol monohydrate in human atheroma at the atomic level

High-resolution electron microscopic investigation of cholesterol monohydrate crystals obtained from human atheroma was carried out for the purpose of characterization of the crystal lattice, demonstration of crystallization processes and identification of crystal disorders. By high-resolution electron microscopy the crystal structures of perfect cholesterol monohydrate crystals were characterized as regular lattice arrays which consisted of stacks of repetitive rod-shaped substructures ca 1.58 nm long and 0.16 nm wide, with the total thickness of bilayered substructures ca 3.36 nm. These substructures were in an end-to-end arrangement of approximately side-to-side parallel packing, with a centre-to-centre spacing ca 0.32 nm. At the atomic level the lattice arrays were made up of regularly spaced rows of dots ca 0.28 nm × 0.16 nm in size. These dots possessed a six-fold ring-like shape, and were arranged in a hexagonal structure with an additional dot in the centre. High-resolution electron microscopic observations of the partially crystallized particles of cholesterol monohydrate showed various stages of cholesterol crystallization, from very small short-ordered segment of lattice arrays to different sized nano- and microcrystallites in the amorphous matrix of the crystals. Furthermore, crystal growth was also demonstrated from detailed examination of the crystal surfaces, the interfaces between the crystals and the boundary structures between the amorphous and crystalline phases. In addition, high-resolution electron microscopy could clearly identify various kinds of crystal defect in the cholesterol monohydrate crystals, including considerable variations of lattice spacings with focal fragmentation of lattice fringes, derangement of atom-sized dots along the lattice fringes and marked alterations of the morphology of atom-sized dots with the vacancies along the lattice arrays. It is hoped that such information obtained from high-resolution electron microscopic observations of the crystalline cholesterol in human atheroma at the atomic or near-atomic level may be helpful by providing a more complete understanding of the pathogenetic mechanisms responsible for the formation, progression and regression of the acellular lipid-rich cores of advanced atherosclerotic plaques.     

Design strategies of sea urchin teeth: structure,composition and micromechanical relations to function.

The teeth of sea urchins comprise a variety of different structural entities, all of which are composed of magnesium-bearing calcite together with a small amount of organic material. The teeth are worn down continuously, but in such a way that they remain sharp and functional. Here we describe aspects of the structural, compositional and micromechanical properties of the teeth of Paracentrotus lividus using scanning electron microscopy, infrared spectrometry, atomic absorption. X-ray diffraction and microindentation. The S-shaped single crystalline calcitic fibres are one of the main structural elements of the tooth. They extend from the stone part to the keel. The diameter of the fibres increases gradually from less than 1 micron at the stone tip to about 20 microns at the keel end, while their MgCO3 contents decrease from about 13 mol% to about 4.5 mol%. Each fibre is coated by a thin organic sheath and surrounded by polycrystalline calcitic discs containing as much as 35 mol% MgCO3. This structure constitutes a unique kind of gradient fibre-reinforced ceramic matrix composite, whose microhardness and toughness decrease gradually from the stone part to the keel. Primary plates are also important structural elements of the tooth. Each primary plate has a very unusual sandwich-like structure with a calcitic envelope surrounding a thin apparently amorphous CaCO3 layer. This central layer, together with the primary plate/disc interface, improves the toughness of this zone by stopping and blunting cracks. The self-sharpening function of the teeth is believed to result from the combination of the geometrical shape of the main structural elements and their spatial arrangement, the interfacial strength between structural elements, and the hardness gradient extending from the working stone part to the surrounding zones. The sea urchin tooth structure possesses an array of interesting functional design features, some of which may possibly be applicable to materials science.     

Measurement of cholesterol gallstone growth in vitro

Methods to study growth of gallstones in the laboratory have not been reported. We here present such a method. Human cholesterol gallstones were harvested from patients with multiple nearly identical stones. The gallstones were washed and added to supersaturated model biles and the formation of cholesterol crystals and the increases in mass of human cholesterol gallstones were studied concurrently, over a period of weeks, using nephelometry and a microbalance, respectively. All stones incubated in model biles supersaturated with cholesterol increased in mass. Increases in the degree of supersaturation of cholesterol in the model biles resulted in increased growth of stones. The mass increases, the growth rates, and the spatial orientation of accreted crystalline cholesterol differed among various stone types. The kinetics and structures of stone growth were similar when the stones were incubated in supersaturated, native, human gallbladder biles. The structure of accreted cholesterol was the same as found on the surface of some human gallstones that were harvested during apparent active growth in situ. This simple method allows accurate measurements of stone growth in vitro, in patterns that mimic stone growth in vivo, and is useful for studies on the relationships of gallstone growth and the kinetics of cholesterol crystallization.     

Plastic deformation behaviours of CuZr amorphous/crystalline nanolaminate: a molecular dynamics study

Abstract

The plastic deformation behaviours of Cu₅₀Zr₅₀/B2 CuZr amorphous/crystalline nanolaminate were studied at the atomic scale using molecular dynamics simulations. For pure metallic glass, the highly localised shear banding leads to the overall shear failure. And the plastic deformation of B2 CuZr crystal is mainly determined by the martensitic phase transformation. The composite material, nanolaminate, achieves great improvement of plastic deformation compared with the pure metallic glass and the pure crystal. This plasticity enhancement is attributed to two mechanisms: the suppression effect of the nucleation and propagation of the shear band and the regulating effect of the distribution of the shear transformation zones and phase transformation zones. The shear transformation zones can be induced by the interaction between phase transformation zones and interface. The immature shear band or shear transformations zones and phase transformation zones form a network to transmit the strain jointly within the entire sample. Amorphous/crystalline interface connects different layers and transmits the strain within the nanolaminate. Interfaces also plays the roles of source and sink of the shear transformation zones, shear band and phase transformation zones. On the basis of this plastic deformation mechanism, the present study provides a route for controlling the plasticity properties of amorphous/crystalline nanolaminate.     

Metastability and transformation of polymorphic crystals in biodegradable poly(butylene adipate)

Polymorphism phenomenon of melt-crystallized poly(butylene adipate) (PBA) has been studied by wide-angle X-ray diffraction (WAXD), small-angle X-ray scattering (SAXS), and differential scanning calorimetry (DSC). It has been found that the isothermal crystallization leads to the formation of PBA polymorphic crystals, simply by changing the crystallization temperature. The PBA alpha crystal, beta crystal, and the mixture of two crystal forms grow at the crystallization temperatures above 32 degrees C, below 27 degrees C, and between these two temperatures, respectively. The relationship between PBA polymorphism and melting behaviors has been analyzed by the assignments of multiple melting peaks. Accordingly, the equilibrium melting temperatures Tm degrees of both alpha and beta crystals were determined by Hoffman-Weeks and Gibbs-Thomson equations for the purpose of understanding the structural metastability. The Tm degrees of the PBA alpha crystal was found to be higher than that of the beta crystal, indicating that the PBA alpha crystal form is a structurally stable phase and that the beta crystal form is a metastable phase. The analysis of growth kinetics of PBA polymorphic crystals indicates that the metastable PBA beta crystal is indeed the kinetically preferential result. Based on the thermal and kinetic results, the phenomenon of stability inversion with crystal size in melt-crystallized PBA was recognized, in terms of the growth mechanisms of PBA alpha and beta crystals and the transformation of beta to alpha crystals. The PBA beta --> alpha crystal transformation takes place at a sufficiently high annealing temperature, and the transformation has been evident to be a solid-solid-phase transition process accompanied by the thickening of lamellar crystals. The molecular motion of polymer chains in both crystalline and amorphous phases has been discussed to understand the thickening and phase transformation behaviors.     

In situ observation of crystal growth for poly[(S)-lactide] by temperature-controlled atomic force microscopy

The crystallization behavior and crystalline morphologies of poly[(S)-lactide] (P[(S)-LA]) in thin films crystallized isothermally at over 160 degrees C were characterized by transmission electron microscopy and atomic force microscopy (AFM). The dendritic crystal and hexagonal crystal were formed in thin film with thicknesses below 30 nm or over 50 nm, respectively. The crystal structures of dendritic and hexagonal crystals were identical, suggesting that the crystalline morphology of P[(S)-LA] is strongly dependent upon the film thickness. In situ observation of the crystal growth in the P[(S)-LA] thin film at 165 degrees C from the melt was carried out by using temperature-controlled AFM equipped with a heating stage. The initial stage of crystallization and development of lamellae were successfully observed during isothermal crystallization at 165 degrees C. The first forming crystal showed the edge-on orientation, and grew to S-shaped edge-on lamellae. Dendritic flat-on crystals were developed from the S-shaped edge-on lamellae. The growth rates of flat-on and edge-on lamellae were almost identical.     

Tubulin Conformation in Zinc-Induced Sheets and Macrotubes

The protein tubulin is the main constituent of microtubules. Previous studies have shown that zinc ions induce the formation of crystalline sheets and macrotubes of tubulin. Both crystal types are suitable for structural studies by electron crystallography. However, crystallographic structural analysis of tubulin has been hampered by limited crystal size and quality and the inability to control crystal polymorphism. We can obtain well-ordered crystals which are grown upon prolonged incubations (up to 24 hr). The presence of NaCl delays the degradation of the crystals, and addition of the protease inhibitor pepstatin improves crystal quality. The crystal form (sheet or macrotube) can be controlled with incubation conditions. The size of the crystals can reach up to 2 μm in width for the sheets and up to 0.5 μm in diameter for the macrotubes. Both crystal types can reach several micrometers in length. Comparison of the projection maps of the two crystal structures shows that adjacent protofilaments in the macrotubes are shifted by about 6 Å relative to their positions in the sheets. Observable changes of to monomer shape appear to allow close interprotofilament contacts to be maintained in both crystal forms. Images of glucose-embedded specimens obtained under these conditions give structural information beyond 4 Å resolution. Merging of high- and low-resolution data allows for unambiguous assignment of monomer boundaries to high-resolution features.     

Matrix macromolecules in hard tissues control the nucleation and hierarchical assembly of hydroxyapatite

Biogenic minerals found in teeth and bones are synthesized by precise cell-mediated mechanisms. They have superior mechanical properties due to their complex architecture. Control over biomineral properties can be accomplished by regulation of particle size, shape, crystal orientation, and polymorphic structure. In many organisms, biogenic minerals are assembled using a transient amorphous mineral phase. Here we report that organic constituents of bones and teeth, namely type I collagen and dentin matrix protein 1 (DMP1), are effective crystal modulators. They control nucleation of calcium phosphate polymorphs and the assembly of hierarchically ordered crystalline composite material. Both full-length recombinant DMP1 and post-translationally modified native DMP1 were able to nucleate hydroxyapatite (HAP) in the presence of type I collagen. However, the N-terminal domain of DMP1 (amino acid residues 1-334) inhibited HAP formation and stabilized the amorphous phase that was formed. During the nucleation and growth process, the initially formed metastable amorphous calcium phosphate phase transformed into thermodynamically stable crystalline hydroxyapatite in a precisely controlled manner. The organic matrix-mediated controlled transformation of amorphous calcium phosphate into crystalline HAP was confirmed by x-ray diffraction, selected area electron diffraction pattern, Raman spectroscopy, and elemental analysis. The mechanical properties of the protein-mediated HAP crystals were also determined as they reflect the material structure. Such understanding of biomolecule controls on biomineralization promises new insights into the controlled synthesis of crystalline structures.     

Ultrastructure of granules and immunocytochemical localization of luciferase in photocytes of fireflies

Photocyte granules are round to oval, 1–2 μm, contain a peripheral dense area and are of three structural types. Type one granules consist of an amorphous matrix, a bundle of 2–12 microtubules and a flask-shaped vacuole. The type two granule is characterized by a large crystal or several smaller crystals embedded in an amorphous matrix with microtubules lined up along the face of the crystal. The type three granule is filled with a large number of thick-walled tubules (40–50 nm o.d.), usually found in bundles of two to four and a few microtubles. Luciferase has been shown to be localized in these photocyte granules by the immunoferritin technique. Ferritin is not localized over microtubules or flask-shaped vacuoles in type one granules but is randomly distributed over the matrix. In type two granules, ferritin is more densely distributed over the crystals and in type three granules over filamentous structures. There is no ferritin over the microtubules. Other parts of photocytes and of light organs are negative for luciferase. Buffer and anticalliphorin incubated sections showed no ferritin in granules.     

Fragments and dust after Holmium laser lithotripsy with or without “Moses technology”: How are they different?

Urinary stones can be readily disintegrated by Holmium:YAG laser (Holmium laser lithotripsy), resulting in a mixture of small stone dust particles, which will spontaneously evacuate with urine and larger residual fragments (RF) requiring mechanical retrieval. Differences between fragments and dust have not been well characterized. Also, it remains unknown how the recently introduced “Moses technology” may alter stone disintegration products. Three complementary analytical techniques have been used in this study to offer an in‐depth characterization of disintegration products after in vitro Holmium laser lithotripsy: stereoscopic microscopy, scanning electron microscopy and Fourier‐transform infrared spectroscopy. Dust was separated from fragments based on its floating ability in saline irrigation. Depending on initial crystalline constituents, stone dust either conserved attributes found in larger RFs or showed changes in crystalline organization. These included conversion of calcium oxalate dihydrate towards calcium oxalate monohydrate, changes in carbapatite spectra towards an amorphous phase, changes of magnesium ammonium phosphate towards a differing amorphous and crystalline phase and the appearance of hydroxyapatite on brushite fragments. Comparatively, “Moses technology” produced more pronounced changes. These findings provide new insights suggesting a photothermal effect occurring in Holmium laser lithotripsy. Figure: Appearance of hydroxyapatite hexagons on stone dust collected after Holmium laser lithotripsy of a brushite stone using “Moses technology.”      

Diversity of the slippery zone microstructure in pitchers of nine carnivorous Nepenthes taxa

Using a cryo-scanning electron microscope, we studied microstructure of the slippery zone in nine Nepenthes taxa. For N. fusca, N. macrophylla, N. mirabilis, N. ventricosa, N. dicksoniana, and N. veitchii, it was examined here for the first time. Three types of the slippery zone were distinguished among the studied taxa: (1) with well-developed crystalline wax coverage, (2) with greatly reduced wax coverage, and (3) without wax crystals. These data were combined with morphometrical measurements of the two pitcher zones primarily relevant to prey catching and retaining: the slippery zone and the peristome. In species with fully developed wax coverage, the slippery zone was longer and the peristome was narrower compared to those with reduced or lacking crystalline wax. We found statistically significant negative correlation between the relative length of the slippery zone and the relative width of the peristome. Based on the analysis of the relationship between the microstructure of the slippery zone and pitcher macromorphology, two main types of pitchers in Nepenthes are proposed: (a) traps based predominantly on the waxy slippery zone and (b) peristome-based traps.     

Calcium Oxalate Crystals in Eucalypt Ectomycorrhizae: Morphochemical Characterization

Ectomycorrhizal fungi are ubiquitous in forest ecosystems, benefitting plants principally by increasing the uptake of water and nutrients such as calcium from the soil. Previous work has demonstrated accumulation of crystallites in eucalypt ectomycorrhizas, but detailed morphological and chemical characterization of these crystals has not been performed. In this work, cross sections of acetic acid-treated and cleared ectomycorrhizal fragments were visualized by polarized light microscopy to evaluate the location of crystals within cortical root cells. Ectomycorrhizal sections were also observed by scanning electron microscopy (SEM) coupled with energy dispersive x-ray (EDS) microprobe analysis. The predominant forms of crystals were crystal sand (granules) and concretions. Calcium, carbon and oxygen were detected by EDS as constituent elements and similar elemental profiles were observed between both crystal morphologies. All analyzed crystalline structures were characterized as calcium oxalate crystals. This is the first report of the stoichiometry and morphology of crystals occurring in eucalypt ectomycorrhizas in tropical soils. The data corroborates the role of ectomycorrhizae in the uptake and accumulation of calcium in the form of calcium oxalate crystals in hybrid eucalypt plants.     

Crystalline structure of poly(hexamethylene adipate). Study on the morphology and the enzymatic degradation of single crystals

The crystalline structure of polyester 6 6 was studied by means of X-ray and electron diffraction and real-space electron microscopy. An orthorhombic unit cell containing eight chain segments with a quasi planar zigzag conformation was derived. The chain axis projection could be defined by a small rectangular cell containing only two molecular segments. Simulation of electron diffraction patterns indicates that molecular segments were arranged with azimuthal angles close to +/-46 degrees . X-ray diffraction patterns suggested that the large dimensions of the unit cell were a consequence of a slight shift between neighboring chains that improved the electrostatic interactions. Chain-folded lamellar crystals were obtained by isothermal crystallization of dilute n-hexanol or n-octanol solutions. The crystalline habit was studied, and the influence of temperature was evaluated. A regular folding surface was observed by using polyethylene decoration techniques. Lamellar crystals were easily degraded with different lipases. A preferential enzymatic attack was, in some cases, observed to occur in the crystal edges, giving rise to highly irregular borders with a fringed texture.     

Scanning electron microscope observations of the organic matrix in the otolith of the teleost fish Fundulus heteroclitus (Linnaeus) and Tilapia nilotica (Linnaeus)

Scanning electron microscope observations of sagitta otoliths of Fundulus heteroclitus (Linnaeus) and Tilapia nilotica (Linnaeus) have revealed that the “discontinuous zone” is a narrow band of organic matrix consisting of fibers ≈900 Å thick, that in turn are composed of thin fibers ≈200 Å thick. The “incremental zone” is the crystalline layer with crystals elongated perpendicular to the otolith periphery that are usually terminated at the discontinuous zones. The crystals are embedded in organic matrix fibers that appear similar to and continuous with the fibers of the discontinuous zones. Frequently, these fibers aggregate into matrix sheets. Based on these findings, a possible process of otolith formation is proposed.     

Ice crystals formed in tissue during cryosurgery. II. Electron microscopy

Tissues frozen by means of a cryosurgical probe have been examined by electron microscopy following techniques designed to preserve the ice crystal spaces.Ice crystals appeared similar whether tissues were quenched or not following cryosurgery and the various techniques of dehydration resulted in similar ice crystal architecture.Ice crystal spaces in the area deep to the freezing probe were intracellular both in epithelium and muscle although in the muscle zone some fibers contained large and others small crystal spaces. It is suggested that this might be due to variations in the local blood supply.At the periphery of the frozen area ice crystals were usually extracellular producing gross distortion of the cells which, however, retained intracellular structural integrity. These results are consistent with the belief of many workers that intracellular ice is lethal while extracellular ice is not, but no evidence of penetration of cell membrane by ice crystals was seen.     

Crystal-associated matrix components in rat incisor enamel

Summary Sections of glutaraldehyde-OsO₄-fixed, plastic-embedded rat incisor enamel were left untreated, stained, decalcifed (1% formic acid in 10% sodium citrate), or decalcified-stained. The presence of apatite crystals was monitored with electron diffraction. After brief decalcification and staining, apatite crystals and matrix components were visualized in the same field. The ghost was continuous with crystal fragments, and the coat appeared as a dense line next to crystals and ghosts. Position of ghosts and crystals at the ameloblast-enamel junction (AEJ) of the secretion zone suggested that there may be a lag of no more than 1/5 min between the elaboration of ghost and crystal. A major change in enamel morphology occurs between the AEJ and the deep enamel of the secretion zone. The ghost becomes thinner, the coat more pronounced, and the crystal enlarges. There is only little change from the deep secretion to the maturation zone enamel.     

Effect of gallbladder hypomotility on cholesterol crystallization and growth in CCK-deficient mice

We investigated the effect of gallbladder hypomotility on cholesterol crystallization and growth during the early stage of gallstone formation in CCK knockout mice. Contrary to wild-type mice, fasting gallbladder volumes were enlarged and the response of gallbladder emptying to a high-fat meal was impaired in knockout mice on chow or the lithogenic diet. In the lithogenic state, large amounts of mucin gel and liquid crystals as well as arc-like and tubular crystals formed first, followed by rapid formation of classic parallelogram-shaped cholesterol monohydrate crystals in knockout mice. Furthermore, three patterns of crystal growth habits were observed: proportional enlargement, spiral dislocation growth, and twin crystal growth, all enlarging solid cholesterol crystals. At day 15 on the lithogenic diet, 75% of knockout mice formed gallstones. However, wild-type mice formed very little mucin gel, liquid, and solid crystals, and gallstones were not observed. We conclude that lack of CCK induces gallbladder hypomotility that prolongs the residence time of excess cholesterol in the gallbladder, leading to rapid crystallization and precipitation of solid cholesterol crystals. Moreover, during the early stage of gallstone formation, there are two pathways of liquid and polymorph anhydrous crystals evolving to monohydrate crystals and three modes for cholesterol crystal growth.     

Aqueous dissolution of crystalline and amorphous amylose-alcohol complexes

Complexes of amylose, the linear starch polysaccharide, with linear alcohols having chain lengths varying from 4 to 8 carbon atoms, were prepared. Either crystalline or amorphous complexes could be formed depending on preparation conditions. Crystalline complexes gave sharp X-ray diffraction patterns, characteristic of the V_H form of amylose, whereas no observable pattern was obtained from the amorphous form. Thermal dissociation of the complexes occurred at increasing temperatures with increasing alcohol chain length. Crystalline complexes dissociated at temperatures approximately 23°C higher than their amorphous counterparts and the enthalpy of dissociation was also greater for the crystalline samples. Enthalpy values were independent of alcohol chain length. Differences in thermal behaviour of the two types of complex may be described in terms of the polymer crystal lattice energy and may explain the variability of reported results for complex dissociation in the literature.     

Structure of tropomyosin at 9 angstroms resolution.

We have used molecular replacement followed by a highly parameterized refinement to determine the structure of tropomyosin crystals to a resolution to 9 A. The shape, coiled-coil structure and interactions of the molecules in the crystals have been determined. These crystals have C2 symmetry with a = 259.7 A, b = 55.3 A, c = 135.6 A and beta = 97.2 degrees. Because of the unusual distribution of intensity in X-ray diffraction patterns from these crystals, it was possible to solve the rotation problem by inspection of qualitative aspects of the diffraction data and to define unequivocally the general alignment of the molecules along the (332) and (3-32) directions of the unit cell. The translation function was then solved by a direct search procedure, while electron microscopy of a related crystal form indicated the probable location of molecular ends in the asymmetric unit, as well as the anti-parallel arrangement. The structural model we have obtained is much clearer than that obtained previously with crystals of extraordinarily high solvent content and shows the two alpha-helices of the coiled coil over most of the length of the molecules and establishes the coiled-coil pitch at 140(+/- 10) A. Moreover, the precise value of the coiled-coil pitch varies along the molecule, probably in response to local variations in the amino acid sequence, which we have determined by sequencing the appropriate cDNA. The crystals are constructed from layers of tropomyosin filaments. There are two molecules in the crystallographic asymmetric unit and the molecules within a layer are bent into an approximately sinusoidal profile. Molecules in consecutive layers in the crystal lie at an angle relative to one another as found in crystalline arrays of actin and myosin rod. There are three classes of interactions between tropomyosin molecules in the spermine-induced crystals and these give some insights into the molecular interactions between coiled-coil molecules that may have implications for assemblies such as muscle thick filaments and intermediate filaments. In interactions within a layer, the geometry of coiled-coil contacts is retained, whereas in contacts between molecules in adjacent layers the coiled-coil geometry varies and these interactions instead appear to be dominated by the repeating pattern of charged zones along the molecule.