Isothermal crystallization of polyhydroxyalkanoate (PHA) utilizing Raman spectroscopy to follow chain packing as well as molecular motion.

Raman spectra of bioplastic poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHx at 13.8 % Hx) were recorded between -250 cm-1 and 3200 cm-1 during isothermal crystallization at 25°C after quenching from the melt in liquid nitrogen. At room temperature the crystallization proceeds slowly, so spectra were recorded over a 14-hour period. While there are spectral changes throughout the spectrum, the focus was on interpretable bands known to be sensitive to crystalline form. These bands included the carbonyl band that sharpens and shifts, a pair of bands on the high energy side of the carbon-hydrogen stretch, and a low frequency band that we assign to the molecular phonon in the crystal unit cell. After appropriate pre-processing of the spectra, they were further analyzed by 2D-COS (two-dimensional correlation spectroscopy) that provides determination of the order in which the polymer functional regions assemble into the crystalline state. According to this analysis one of the methyl CH's interacts with the carbonyl bond to produce a line at 3000 cm-1. Following that, multiple changes appear in the carbonyl region, the strong CH band at 2930 cm-1 of the crystalline phase grows, then the 80 cm-1 phonon band, and the splitting of the methyl CH only appears after the phonon. From this sequence one can derive a picture of how the polymer unit locks into the crystal form. This can be of interest to commercialization of the materials because mechanical properties are intimately controlled by the crystallinity of the material. By understanding how the crystallization process proceeds, it can be engineered to be "fit for purpose" for a polymer targeted for a specific use.

Optimizing depth resolution in confocal Raman microscopy: a comparison of metallurgical, dry corrected, and oil immersion objectives.

Spherical aberration is probably the most important factor limiting the practical performance of a confocal Raman microscope. This paper suggests some simple samples that can be readily fabricated in any laboratory to test the performance of a confocal Raman microscope under realistic operating conditions (i.e., a deeply buried interface, rather than the often-selected alternative of a bare silicon wafer or a thin film in air). The samples chosen were silicon wafers buried beneath transparent polymeric or glass overlayers, and a polymer laminate buried beneath a cover glass. These samples were used to compare the performance of three types of objectives (metallurgical, oil immersion, and dry corrected) in terms of depth resolution and signal throughput. The oil immersion objective gave the best depth resolution and intensity, followed by a dry corrected (60x, 0.9 numerical aperture) objective. The 100x metallurgical objective was the worst choice, with degradations of approximately 5x and 8x in the depth resolution and signal from a silicon wafer, comparing a bare wafer with one buried under a 150 microm cover glass. In particular, the high signal level obtained makes the immersion objective an attractive choice. Results from the buried laminate were even more impressive; a 30x improvement in spectral contrast was obtained using the oil immersion objective to analyze a thin (19 microm) coating on a PET substrate, buried beneath a 150 microm cover glass, compared with the metallurgical objective.

Confocal spectral imaging in tissue with contrast provided by Raman vibrational signatures.

Confocal Raman imaging of biological materials offers the opportunity to extract chemical information on histologically defined regions and on sub-cellular organelles. This article reviews the technology and some successful applications. The chemical contrast from vibrational Raman spectroscopy is derived from the specific atomic motion of every molecule as detected by the Raman phenomenon. Examples show the successful identification of foreign material in pathological specimens, identification of lipid-type and calcium mineral-type in a mouse model of atherosclerosis, and component mapping in a pharmaceutical tablet. It is suggested that these methods can even be useful in studying metabolic disorders.

Age-related changes in physicochemical properties of mineral crystals are related to impaired mechanical function of cortical bone.

The measures of bone mass and architecture need to be supplemented with physicochemical and compositional measures for better assessment of fracture risk. In the current studies, we investigated the effects of physicochemical properties of mineral crystals on tissue and organ-level mechanical function of aging rat cortical bone. Our hypothesis was that age-related changes in physicochemical properties of mineral crystals are related to impaired elastic deformability of cortical bone tissue. Raman microspectroscopy was used to investigate the age-related changes in mineralization (relative amounts of mineral and organic matrix), the substitution of carbonate ions in phosphate positions (type-B carbonate substitution) and mineral crystallinity (the orderliness of crystal lattice) of femurs from young adult (3-month old), middle-aged (8-month old) and aged (24-month old) female Sprague-Dawley rats. Cross-sectional properties, the area and the moment of inertia at the mid-diaphysis, were histomorphometrically quantified and the elastic deformation capacity of femurs was quantified via three-point bending tests. It was observed that the elastic deformation capacity of aged rats was significantly impaired both at the tissue and the organ levels with increasing age. In parallel with this impairment in the elastic deformability and in support of our hypothesis, we found that increasing mineralization, increasing crystallinity and increasing type-B carbonate substitution were significantly correlated with decreasing elastic deformation capacity with age. We conclude that the measure of bone mass needs to be supplemented with measures reflecting the physicochemical status of mineral crystals for improved assessment of fracture susceptibility.

Characterization and application of Raman labels for confocal Raman microspectroscopic detection of cellular proteins in single cells.

A method using confocal Raman microspectroscopy for the detection of cellular proteins in single intact cells was developed. Two approaches were used to improve the detection of these cellular components. First, compounds with high Raman scattering were investigated for potential use as Raman labels. Raman labels were conjugated to either biomolecules or biotin and used as markers in the detection of cellular enzymes and receptors. Second, silver colloids were used to increase the surface-enhanced Raman scatter (SERS) of these Raman labels. Cresyl violet and dimethylaminoazobenzene are Raman labels that provide very sensitive SERS detection by a confocal Raman microscope with a HeNe laser at wavelength of 632.8 nm. The detection of 12-lipoxygenase and cyclooxygenase-1 in single bovine coronary artery endothelial cells and the binding of angiotensin II to its receptors in zona glomerulosa cells was demonstrated.

Aging of microstructural compartments in human compact bone.

Composition of microstructural compartments in compact bone of aging male subjects was assessed using Raman microscopy. Secondary mineralization of unremodeled fragments persisted for two decades. Replacement of these tissue fragments with secondary osteons kept mean composition constant over age, but at a fully mineralized limit. Slowing of remodeling may increase fracture susceptibility through an increase in proportion of highly mineralized tissue. In this study, the aging process in the microstructural compartments of human femoral cortical bone was investigated and related to changes in the overall tissue composition within the age range of 17-73 years. Raman microprobe analysis was used to assess the mineral content, mineral crystallinity, and carbonate substitution in fragments of primary lamellar bone that survived remodeling for decades. Tissue composition of the secondary osteonal population was investigated to determine the composition of turned over tissue volume. Finally, Raman spectral analysis of homogenized tissue was performed to evaluate the effects of unremodeled and newly formed tissue on the overall tissue composition. The chemical composition of the primary lamellar bone exhibited two chronological stages. Organic matrix became more mineralized and the crystallinity of the mineral improved during the first stage, which lasted for two decades. The mineral content and the mineral crystallinity did not vary during the second stage. The results for the primary lamellar bone demonstrated that physiological mineralization, as evidenced by crystal growth and maturation, is a continuous process that may persist as long as two decades, and the growth and maturation process stops after the organic matrix becomes "fully mineralized." The average mineral content and the average mineral crystallinity of the homogenized tissue did not change with age. It was also observed that the mineral content of the homogenized tissue was consistently greater than the osteons and similar to the "fully mineralized" stage of primary bone. The results of this study demonstrated that unremodeled compartments of bone grow older through maturation and growth of mineral crystals in a protracted fashion. However, the secondary osteonal remodeling impedes this aging process and maintains the mean tissue age fairly constant over decades. Therefore, slowing of remodeling may lead to brittle bone tissue through accumulation of fully mineralized tissue fragments.