A combination of two-photon fluorescence (TPF), second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) imaging has been used to investigate the elastin fibre network in healthy equine articular cartilage from the metacarpophalangeal joint. The elastin fibres were identified using their intrinsic twophoton fluorescence and immuno-staining was used to confirm the identity of these fibres. SHG was used to reveal the collagen matrix and the collagen fibre orientations were determined from their SHG polarization sensitivity, while CARS was used to clearly delineate the cell boundaries. Extensive elastin fibre networks were found in all the joint regions investigated. The elastin was found predominantly in the superficial zone (upper 50 lm) and was aligned parallel to the articular surface. Elastin was also detected in the pericellular matrix surrounding the superficial chondrocytes; however, individual fibres could not be resolved in this region. Variations in the density and organization of the fibres were observed in different regions on the joint surface.

Since its invention in the mid 1980s atomic force microscopy has revolutionised the way in which surfaces can be imaged. Close to atomic resolution has been achieved for some materials and numerous images of molecules on surfaces have been recorded. Atomic force microscopy has also been of benefit to biology where protein molecules on surfaces have been studied and even whole cells have been investigated. Here we report a study of red blood cells which have been imaged in a physiological medium. At high resolution, the underlying cytoskeleton of the blood cell has been resolved and flaws in the cytoskeleton structure may be observed. Comparison of the normal 'doughnut' shaped cells with swollen cells has been undertaken. Differences in both the global properties of the cells and in the local features in cytoskeleton structure have been observed.

No abstract

Raman microspectroscopy has been used to investigate the structure of alpha-elastin and fibrous elastin from ligament and aorta, and to explore changes associated with mechanical strain and temperature. Although no vibrational modes associated with cross-linking of the fibers could be identified, the secondary structure of dehydrated fibrous elastin was significantly different from alpha-elastin. The former differed from previous experimental measurements, but was close to the theoretical predictions with 36% beta-structures, 46% unordered, and 18% alpha-helix. Alpha-elastin contained 29% beta-structures, 53% unordered, and 18% alpha-helix. In nuchal fibers the amide I mode was polarized, consistent with the peptide bond. Strains of up to 60% in ligament fiber bundles resulted in no significant shifts in peak position or in secondary structure. Polarization measurements revealed that the peptide bonds and several side chains re-orientated closer to the fiber axis. Heating nuchal fibers to 60 degrees C to increase the energetic component of the elasticity was associated with a 30% increase in the proportion of beta-structures in the amide I band, a 50% increase in the amide III band, and a 50% reduction in the signal from bound water.

The complex structure of the annulus fibrosus is strongly related to its mechanical properties. Recent work showed that it is possible to observe the relative movement of fibre bundles in loaded cow tail annulus; the aim of this work was to describe and quantify annulus fibrosus micromechanics in degenerated human disc, and compare it with cow tail annulus, an animal model often used in the literature. Second harmonic generation was used to image the collagen matrix in twenty strips of annulus fibrosus harvested from intervertebral disc of seven patients undergoing surgery. Samples were loaded to 6% tensile strain in 1% steps. Elastic modulus was calculated from loading curves, and micromechanical strains were calculated from the images using custom software. The same protocol was applied to twenty strips of annulus harvested from cow tail discs. Significant morphological differences were found between human and cow tail samples, the most striking being the lack of collagen fibre crimp in the former. Fibres were also observed bending and running from one lamella to the other, forming a strong flexible interface. Interdigitation of fibre bundles was also present at this interface. Quantitative results show complex patterns of inter-bundle and inter-lamellar behaviour, with inter-bundle sliding being the main strain mechanism. Elastic modulus was similar between species, and it was not affected by the degree of degeneration. This work gives an insight into the complex structure and mechanical function of the annulus fibrosus, which should be accounted for in disc numerical modelling.Electronic supplementary materialThe online version of this article (doi:10.1007/s10237-017-0900-z) contains supplementary material, which is available to authorized users.

No abstract

No abstract

Raman spectroscopy is a well‐established technique that allows both chemical and structural analysis of materials. Raman spectra are often complex and extracting meaningful information is easily hindered by spectral interferences; one of the most significant sources being variations in background. Raman spectra have diverse sources of background making it hard to eliminate them or theoretically to predict the form of the baseline, which frequently varies between samples. Although many different methods for baseline removal have been proposed, most require some form of user input. User input is also subjective and consequently less reproducible than automated methods and variations in baseline subtraction can distort peak heights leading to erroneous results. We present a Bayesian Whittaker–Henderson smoother for spectral baseline estimation and peak extraction. It is a generalisation of the Whittaker–Henderson smoother, a regularised regression algorithm. We introduce hierarchical priors for model parameters of the smoother and propose a global aligner for consistent peak extraction across multiple spectra. We show that this novel smoother significantly outperforms several existing smoothers. Copyright © 2012 John Wiley & Sons, Ltd.