Copyright (c) 2013 Dublin Institute of Technology All rights reserved. http://arrow.dit.ie/biophonart Recent documents in Articles en-us Wed, 29 May 2013 12:05:51 PDT 3600 http://arrow.dit.ie/biophonart/4 http://arrow.dit.ie/biophonart/4 Mon, 22 Apr 2013 01:55:10 PDT Raman and Infrared absorption spectroscopies are compared for the analysis of human hand skin tissue sections. The tissue sections have been formalin fixed and paraffin processed, and subsequently dewaxed. Fourier Transform infrared (FTIR) spectra are preprocessed using the resonant Mie – extended multiplicative scattering algorithm to remove spectral artefacts. FTIR images of resolution 4cm^-1, analysed using K-means cluster analysis, reveal the double layer structure of the dermis and epidermis, but no further layer differentiation is achieved using the higher spatial resolution of the Attenuated Total Reflection imaging or improved spectral resolution of 2cm^-1. At comparable spectral and spatial resolutions and measurement on the same samples, Raman scattering produces spectra of significantly higher spectral detail and can differentiate the stratum corneum from the underlying epithelial layer, and, in the absence of melanin in an artificial skin model, can further differentiate the basal layer from the overlying epithelium. The differences in the performance of the techniques are therefore not instrumentational and are discussed in terms of the technological and fundamental differences between the two complementary techniques.

]]> Syed M. Ali et al. http://arrow.dit.ie/biophonart/3 http://arrow.dit.ie/biophonart/3 Fri, 11 Jan 2013 02:30:20 PST Raman spectroscopy coupled with K-means clustering analysis (KMCA) is employed to elucidate the biochemical structure of human skin tissue sections, and the effects of tissue processing. Both hand and thigh sections of human cadavers were analysed in their unprocessed and formalin fixed paraffin processed (FFPP) and subsequently dewaxed forms. In unprocessed sections, KMCA reveals clear differentiation of the stratum corneum, intermediate underlying epithelium and dermal layers for sections from both anatomical sites. The stratum corneum is seen to be relatively rich in lipidic content; the spectrum of the subjacent layers is strongly influenced by the presence of melanin, while that of the dermis is dominated by the characteristics of collagen. For a given anatomical site, little difference in layer structure and biochemistry is observed between samples from different cadavers. However, the hand and thigh sections are consistently differentiated for all cadavers, largely based on lipidic profiles. In dewaxed FFPP samples, while the stratum corneum, intermediate and dermal layers are clearly differentiated by KMCA of Raman maps of tissue sections, the lipidic contributions to the spectra are significantly reduced, with the result that respective skin layers from different anatomical sites become indistinguishable. While efficient at removing the fixing wax, the tissue processing also efficiently removes the structurally similar lipidic components of the skin layers. In studies of dermatological processes in which lipids play an important role, such as wound healing, dewaxed samples are therefore not appropriate. Removal of the lipids does however accentuate the spectral features of the cellular and protein components, which may be more appropriate for retrospective analysis of disease progression and biochemical analysis using tissue banks.

]]> Syed Mehmood Ali et al. http://arrow.dit.ie/biophonart/2 http://arrow.dit.ie/biophonart/2 Fri, 04 Jan 2013 04:15:14 PST Raman microspectroscopy is widely used for molecular characterisation of tissue samples. Nevertheless, when working in vitro on tissue sections, the presence of a broad background to the spectra remains problematic and its removal requires advanced methods for pre-processing of the data. To date, research efforts have been primarily devoted to development of techniques of statistical analysis to extract the relevant information contained in the spectra. However, few attempts have been made to understand the origin of the background and to improve the protocols used for the collection of Raman spectra that could lead to the reduction or elimination of the background. It has been demonstrated that measurement at 785nm in water immersion significantly reduces the Raman background of both pure biochemical components and tissue sections, associating the background at 785nm with a scattering phenomenon rather than fluorescence. It is however of interest to probe the dependence of the observed background and any time evolution normally associated with photobleaching of fluorophores, under dry and immersed conditions, on the source wavelength. Using 785nm or 660nm as source, extended exposure of dried skin tissue sections to the laser results in a time dependent reduction of the background present in the Raman spectra. When working in water immersion, the overall background as well as the evolution over time is greatly reduced and the background is seen to stabilise after ~20 seconds exposure. Using 532 nm or 473 nm as source for the examination of dried tissue sections, visible photodamage of the sample limits the laser power usable for the collection of spectra to 5 mW. Immersion of the tissue sections protects against photodamage and laser powers of up to 30 mW can be used without any visible damage. Under these conditions, the background is significantly reduced and good quality Raman spectra can be recorded. By adapting the protocol usually used for the collection of Raman spectra, this study clearly demonstrates that other approaches rather than mathematical manipulation of the data can be used to deal with the intrinsic background commonly observable. Notably, the dependence of the background and its time evolution under prolonged exposure on sample environment potentially sheds light on its origin as due to sample morphology (scattering) rather than chemical content (fluorescence). Overall, the study demonstrates that, in addition to reduced background, the photostability of the samples is significantly enhanced in an immersion geometry.

]]> Franck Bonnier et al. http://arrow.dit.ie/biophonart/1 http://arrow.dit.ie/biophonart/1 Mon, 20 Feb 2012 01:35:04 PST Raman spectroscopy is employed to determine the suitability of the U20S osteoblast-like cell line for use as a model for human primary osteoblasts, with emphasis on the ability of these cell types to replicate their tissue of origin. It was found that both cell types demonstrated early stage mineral deposition that followed significantly different growth patterns. Analysis of the growth pattern and spectral data from primary cells revealed increasing bone quality ratios and a high crystallinity, consistent with previous reports. Conversely the investigation of the U20S osteoblast-like cell line provided evidence of dense multilayered mineralised regions that corresponded more closely to native bone in terms of its crystallinity and bone quality ratios. This finding contradicts previous reports on U20S osteoblast-like cells which have consistently described them as non-osteoinductive when cultured in various conditions on a number of substrates. This work demonstrates the successful application of Raman spectroscopy combined with biological and multivariate analysis for the investigation of osteoblast-like U20S cells and human primary osteoblasts, specifically with focus on the osteoinductive ability of the osteoblast-like cell line and the comparative differences in relation to the primary osteoblasts.

]]> Lindsay McManus et al.