mekhala's library 69 articles

Abstract

On account of its sensitivity to chirality, Raman optical activity (ROA), which may be measured as a small difference in the intensity of vibrational Raman scattering from chiral molecules in right- and left-circularly polarized incident light, or as the intensity of a small circularly polarized component in the scattered light, is a powerful probe of the structure of biomolecules. Protein ROA spectra provide information on secondary and tertiary structures of polypeptide backbones, backbone hydration and side-chain conformations, and on structural elements ...

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The Raman spectra of poly-L-hydroxyproline in the solid state and aqueous solution have been obtained. From the spectra, the conformations of the polypeptide in the solid and aqueous solution are found to be the same. Major differences in the spectra between the solid and solution are traced to environmental influences of the pyrrolidine group as indicated by studies of the monomers. The Raman spectra of the imino acids, proline and hydroxyproline, as the dipolar ions, the hydrochlorides, and the sodium salts ...

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Osteogenesis imperfecta (OI) is a heritable disease characterized by skeletal deformities and brittle bones. In the current study, the nature of the mineral in long bones of a mouse model of OI (oim/oim, a mutant which produces an alpha 1(I) collagen homotrimer) was examined by Fourier transform infrared microscopy. The mineral:matrix ratio of oim/oim cortical bone was greater than that of the heterozygous oim/+ and of the normal +/+ bones, probably as a result of reduced collagen content. The molecular environments ...

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Research on fluorescent semiconductor nanocrystals (also known as quantum dots or qdots) has evolved over the past two decades from electronic materials science to biological applications. We review current approaches to the synthesis, solubilization, and functionalization of qdots and their applications to cell and animal biology. Recent examples of their experimental use include the observation of diffusion of individual glycine receptors in living neurons and the identification of lymph nodes in live animals by near-infrared emission during surgery. The new generations ...

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There are an increasing number of instrumental methods for obtaining data from biochemical processes, many of which now provide information on many (indeed many hundreds) of variables simultaneously. The wealth of data that these methods provide, however, is useless without the means to extract the required information. As instruments advance, and the quantity of data produced increases, the fields of bioinformatics and chemometrics have consequently grown greatly in importance. The chemometric methods nowadays available are both powerful and dangerous, and there ...

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Bone loss due to trauma or disease is an increasingly serious health problem. Current clinical treatments for critical-sized defects are problematic and often yield poor healing due to the complicated anatomy and physiology of bone tissue, as well as the limitations of medical technology. Bone tissue engineering offers a promising alternative strategy of healing severe bone injuries by utilizing the body's natural biological response to tissue damage in conjunction with engineering principles. Osteogenic cells, growth factors, and biomaterial scaffolds form the ...

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Polarization sensitive coherent anti-Stokes Raman scattering (PCARS) spectroscopy is a fruitful technique to study Raman vibrations of diluted molecules under off-electron resonant conditions. We apply PCARS as a direct spectroscopic method to investigate the broad amide I band of proteins in heavy water. In spontaneous Raman spectroscopy, this band is not well resolved. We fit a number of spectra taken of each protein under different polarization conditions, with a single set of parameters. It then appears that some substructure is observed ...

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Raman scattering from membranes, cells, and tissues must all be anisotropic, because the molecular orientations in these biological systems are anisotropic. How can such observed Raman scattering anisotropy be related with a biologically relevant molecular arrangement? This question is the subject of this paper. A general method of addressing this question will be given, with three examples illustrating the use of the method: (1) carotenoid arrangement in the eyespot of Chlamydomonas, (2) orientation of the tryptophan side chain in the coat ...

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This investigation determined the relative importance of collagen fiber orientation, porosity, density, and mineralization in determining the tensile strength of bovine cortical bone. Thirty-nine specimens were tested for failure stress and the values of eight histologic and compositional parameters: collagen fiber orientation, wet and dry apparent density, percent mineralization of the bone matrix, and several components of porosity (Haversian canals, Volkmann's canals, and plexiform vascular spaces). Linear regression analysis showed that collagen fiber orientation was consistently the single best predictor of ...

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Existing dynamic mechanical testing systems are not well-suited for Raman spectroscopic study of the response of bone tissue chemical structure to mechanical loading. This paper describes design and validation of a testing system for this application. The body of the apparatus is waterproof and is closed to allow passive maintenance of tissue hydration. Specialized specimen grips for tension/compression and for bending studies are described. The system is shown to provide reproducible data on test specimens of murine femora and radii. Stability ...

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Raman microspectroscopy is widely used for musculoskeletal tissues studies. But the fluorescence background obscures prominent Raman bands of mineral and matrix components of bone tissue. A 532-nm laser irradiation has been used efficiently to remove the fluorescence background from Raman spectra of cortical bone. Photochemical bleaching reduces over 80% of the fluorescence background after 2 h and is found to be nondestructive within 40 min. The use of electron multiplying couple charge detector (EMCCD) enables to acquire Raman spectra of bone ...

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Although healthy dentin is invariably hydrated in vivo, from a perspective of examining the mechanisms of fracture in dentin, it is interesting to consider the role of water hydration. Furthermore, it is feasible that exposure to certain polar solvents, e.g., those found in clinical adhesives, can induce dehydration. In the present study, in vitro deformation and fracture experiments, the latter involving a resistance-curve (R-curve) approach (i.e., toughness evolution with crack extension), were conducted in order to assess changes in the constitutive ...

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We report the use of spatial arrays of single quantum dots (QD) as fluorescent probes to quantify deformations and displacements of bone tissue components (e.g. collagen and carbonated apatite) at the nanometer to micrometer level under mechanical load. Quantum dot bright emission and robustness allow nanometer localization and motion tracking by center of gravity (COG) analysis. Coupons of milled cortical bone are loaded in a purpose-built dynamic mechanical loading system that fits on a microscope stage. We used QD streptavidin conjugates ...

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The age-related deterioration in bone quality and consequent increase in fracture incidence is an obvious health concern that is becoming increasingly significant as the population ages. Raman spectroscopy with deep-ultraviolet excitation (244 nm) is used to measure vibrational spectra from human cortical bone obtained from donors over a wide age range (34-99 years). The UV Raman technique avoids the fluorescence background usually found with visible and near-infrared excitation and, due to resonance Raman effects, is particularly sensitive to the organic component ...

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Raman spectroscopy and imaging are known to be valuable tools for the analysis of bone, the determination of protein secondary structure, and the study of the composition of crystalline materials. We have utilized all of these attributes to examine how mechanical loading and the resulting deformation affects bone ultrastructure, addressing the hypothesis that bone spectra are altered, in both the organic and inorganic regions, in response to mechanical loading/deformation. Using a cylindrical indenter, we have permanently deformed bovine cortical bone specimens ...

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Abstract: Raman spectroscopic markers have been determined for fatigue-related microdamage in bovine bone. Microdamage was induced using a cyclic fatigue loading regime. After loading, the specimens were stained en-bloc with basic fuchsin to facilitate damage visualization and differentiate fatigue-induced damage from cracks generated during subsequent histological sectioning. Bone tissue specimens were examined by light microscopy and hyperspectral near-infrared Raman imaging microscopy. Three regions were defined-tissue with no visible damage, tissue with microcracks, and tissue with diffuse damage. Raman transects, lines of ...

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Bone is a hierarchically structured material with remarkable mechanical performance which may serve as a model for the development of biomimetic materials. Understanding its properties is essential for the assessment of diseases such as osteoporosis. This will lead to a critical evaluation of current therapies and aid in their more targeted development. While the full hierarchical structure of bone is extremely complex and variable, its basic building block, the mineralized collagen fibril, is rather universal. Due to the progress in experimental ...

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Bone is a composite material with a hierarchical structure. Its strength depends on its structural and material properties. In the present study, Raman microspectroscopic and Imaging analyses were employed to study 12 osteons in tissue sections from the femoral midshaft of a healthy human female, with a spatial resolution of approximately 1mum. Spatial changes in amount of mineral and organic matrix, as well as the variation in the mineral content were determined, imaged, and plotted as a function of the polarization ...

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A new technique based on polarized Raman spectroscopy is demonstrated for detecting early dental caries on extracted human teeth. Sound tooth enamel exhibited strong Raman polarization anisotropy whereas early caries consistently showed a lower degree of Raman polarization anisotropy. In particular, for sound enamel, the Raman peak arising from the symmetric nu1 vibration of PO4 3- at 959 cm-1 is strongly polarized. This is in contrast to the spectra of carious lesions that displayed weaker polarization dependence at 959 cm-1. Such ...

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New diagnostic tools are needed for the characterization of dental caries in the early stages of development. If carious lesions are detected early enough, they can be arrested without the need for surgical intervention. The objective of this study was to demonstrate that polarization sensitive optical coherence tomography (PS-OCT) can be used for the imaging of early caries lesions and for the monitoring of lesion progression over time. High-resolution polarization resolved images were acquired of natural caries lesions and simulated caries ...

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Single crystals of synthetic hydroxyapatite have been examined by orientational micro-Raman spectroscopy. The observed Raman bands include the PO4(3-)/OH-internal and external modes over the spectral range from 180 to 3600 cm-1. The Raman-active symmetry tensors (A, E1, and E2) of crystal-class C6 have been assigned to these bands. Intensities of the Raman bands are invariant to the orientation of the a- or b-axis of a single crystal, but depend solely on the c-axis orientation. Changes in the spectral profile thus reflect ...

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Bone exhibits positive form birefringence dominated by and dependent upon the orientation of its collagen. The biomechanical efficacy of bone as a tissue is largely determined by collagen fibers of preferred orientation and distribution (and corresponding orientation of mineral crystallites), and evidence is accumulating to demonstrate that this efficacy extends to function at the organ level. This study has three aims. The first is to provide a Background to the study of circularly polarized light (CPL) investigations of collagen fiber orientation ...

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The composite structure of secondary osteon lamellae, key micro-mechanical components of human bone, has intrigued researchers for the last 300 years. Scanning confocal microscopy here for the first time systematically quantifies collagen orientations by location within the lamellar thickness. Fully calcified lamellar specimens, extinct or bright in cross-section under circularly polarized light, were gently flattened, and then examined along their thickness direction, the radial direction in the previously embedding osteon. Collagen orientation was measured from confocal image stacks. So-called extinct lamellae ...

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This investigation presents new insights into the structure of human secondary lamellae. Lamellar specimens that appear dark and bright on alternate osteon transverse sections under circularly polarizing light were isolated using a new technique, and examined by polarizing light microscopy, synchrotron X-ray diffraction, and confocal microscopy. A distribution of unidirectional collagen bundles and of two overlapping oblique bundles appears on circularly polarizing light microscopy images in relation to the angle between the specimen and the crossed Nicols' planes. The unidirectional collagen ...

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Band-target entropy minimization (BTEM) has been applied to extraction of component spectra from hyperspectral Raman images. In this method singular value decomposition is used to calculate the eigenvectors of the spectroscopic image data set. Bands in non-noise eigenvectors that would normally be used for recovery of spectra are examined for localized spectral features. For a targeted (identified) band, information entropy minimization or a closely related algorithm is used to recover the spectrum containing this feature from the non-noise eigenvectors, plus the ...

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Several methods for determination of the secondary structure of proteins by spectroscopic measurements are reviewed. Circular dichroism (CD) spectroscopy provides rapid determinations of protein secondary structure with dilute solutions and a way to rapidly assess conformational changes resulting from addition of ligands. Both CD and Raman spectroscopies are particularly useful for measurements over a range of temperatures. Infrared (IR) and Raman spectroscopy require only small volumes of protein solution. The frequencies of amide bands are analyzed to determine the distribution of ...

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Despite numerous studies of human biominerals, some problems still remain concerning the relationship between their composition and their structure. For a better understanding of this problem, full spectra of the internal vibrations of human tooth enamel crystallites were obtained through polarized Raman microspectrometry and these are published for the first time. The micro-Raman technique is nondestructive and enables micrometric-scale examination of all the samples with a minimum of artifacts. The spectra show some variation from the predicted bands and many similarities ...

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Raman spectroscopy is used as a probe of ultrastructural (molecular) changes in both the mineral and matrix (protein and glycoprotein, predominantly type I collagen) components in real time of murine cortical bone as it responds to elastic deformation. Because bone is ia composite material, its mechanical properties are dependent on the structure and composition at a variety of dimensional scales. At the ultrastructural level, crystal structure and protein secondary structure distort as the tissue is loaded. These structural changes are followed ...

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The FT-Raman spectra and the pressure dependence of the infrared spectra of the hydrated and anhydrous forms of dicalcium phosphate, CaHPO4.2H2O and CaHPO4, have been studied. The hydrated salt exhibits a phase transition at 21 kbar (1.0 kbar=0.1 Gpa) but no high pressure transition was observed for anhydrous dicalcium phosphate. The O-H stretching frequencies of the water molecules in CaHPO4.2H2O all showed negative pressure dependences and correlate with the O...O distances. The PO-H stretch increased with increasing pressure, indicating a strong ...

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Micro Raman spectroscopy (MRS) can be used to measure strain in crystalline and semicrystalline materials with μm scale spatial resolution. It has been employed as a tool for measuring micromechanical behaviour in composites, and has contributed significantly to understanding of composite behaviour. An overview of the fundamentals of Raman spectroscopy and strain measurements using Raman spectroscopy as well as the instrumentation required is given. Studies using MRS to gain insight into the structure, elastic moduli, and failure modes of several types ...

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The secondary osteon — a fundamental building block in compact bone — is a multilayered cylindrical structure of mineralized collagen fibrils arranged around a blood vessel. Functionally, the osteon must be adapted to the in vivo mechanical stresses in bone at the level of its microstructure. However, questions remain about the precise mechanism by which this is achieved. By application of scanning x-ray diffraction with a micron-sized synchrotron beam, along with measurements of local mineral crystallographic axis direction, we reconstruct the ...

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Summary Ultrathin sections of decalcified human compact bone, observed by transmission electron microscopy, reveal that collagen fibrils can be distributed in the form of a superimposed series of nested arcs. This characteristic pattern has never been interpreted in previous works on compact bone structure. We demonstrate, by goniometric observations at the ultrastructural level, that such series of nested arcs are a consequence of the “twisted plywood” architecture of collagen fibrils in the compact bone matrix. In the same specimens, an ...

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This study is concerned with the cryogenic preservation of intrafibrillar apatite distribution in type I collagen of turkey leg tendons. Cryogenic specimen preparations by the rapid freezing of nonfixed and noncryoprotected leg tendons were performed by two different protocols: (1) low temperature substitution, fixation and staining followed by low temperature embedment; (2) frozen hydrated and air-dried cryosections were examined with the electron microscope at -165 degrees C and normal operating temperatures, respectively. These protocols revealed the axial periodicity for mineralized collagen ...

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Turkey leg tendons at an early stage of mineralization have been thin sectioned and imaged by electron microscopy. At this stage collagen-associated mineral apatite was found to be present within both the gap and overlap zones. The earliest apatite occurs in a microcrystalline form which gives a rather generalized and characteristic density to both the gap and overlap zones; with subsequent development larger defined apatite crystals arise which span gap/overlap zones. Fourier transformation of such images revealed the major 67 nm ...

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Transmission electron micrographs of individual mineralized collagen fibrils show that hydroxyapatite crystals are located mainly within the fibrils at the level of the gap regions. The plate-shaped crystals are observed to be more or less uniformly stacked across the fibril diameter. We therefore suggest that the crystals are primarily located in 'grooves' created by contiguous adjacent gaps. The proposal is consistent with the observed crystal distribution in the fibril and with their average widths, which are almost 10-times greater than an ...

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The organization of apatite crystals and collagen fibrils in mineralized turkey tendon has been studied by electron microscopy and electron diffraction. To minimize artifactual distortions the tissue was examined, for the first time, as isolated fibrils in an aqueous environment of vitreous ice, as well as in conventionally prepared sections. The electron micrographs show that the plate-shaped apatite crystals are arranged in parallel arrays across the collagen fibrils. This provides direct evidence for highly asymmetric assembly in collagen fibrils, and, indeed, ...

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Bone collagen cross-links are now widely used to assess bone resorption levels in many metabolic bone diseases. The post-translational modifications of bone and other mineralizing collagens are significantly different from those of other type I collagen matrices, a fact that has been exploited during recent advances in the development of biochemical markers of bone resorption. The enzymatic collagen cross-linking mechanism is based upon aldehyde formation from specific telopeptide lysine or hydroxylysine residues. The immature ketoimine cross-links in bone form via the ...

Abstract

Using laser Raman microprobe spectroscopy, we have characterized the degree of hydroxylation and the state of atomic order of several natural and synthetic calcium phosphate phases, including apatite of biological (human bone, heated human bone, mouse bone, human and boar dentin, and human and boar enamel), geological, and synthetic origin. Common belief holds that all the studied phases are hydroxylapatite, i.e., an OH-containing mineral with the composition Ca10(PO4)6(OH)2. We observe, however, that OH-incorporation into the apatite crystal lattice is reduced for ...

Abstract

Hydrogen-bearing species in the bone mineral environment were investigated using solid-state NMR spectroscopy of powdered bone, deproteinated bone, and B-type carbonated apatite. Using magic-angle spinning and cross-polarization techniques three types of structurally-bound water were observed in these materials. Two of these water types occupy vacancies within the apatitic mineral crystal in synthetic carbonated apatite and deproteinated bone and serve to stabilize these defect-containing crystals. The third water was observed at the mineral surface in unmodified bone but not in deproteinated bone, ...

Abstract

Studies of the apatitic crystals of bone and enamel by a variety of spectroscopic techniques have established clearly that their chemical composition, short-range order, and physical chemical reactivity are distinctly different from those of pure hydroxyapatite. Moreover, these characteristics change with aging and maturation of the bone and enamel crystals. Phosphorus-31 solid state nuclear magnetic resonance (NMR) spin-spin relaxation studies were carried out on bovine bone and dental enamel crystals of different ages and the data were compared with those obtained ...