In this paper we present an algorithm for analysing sets of FTIR microscopic images of tissue sections. The proposed approach allows one to investigate sets of many FTIR tissue images both with respect to sample information (variation from image to image) and spatial variations of tissues (variation within the image). The algorithm is applied to FTIR microscopy images of beef loin muscles containing myofibre and connective tissue regions. The FTIR microscopy images are taken of sub-samples from five different beef loin muscles that were aged for four different lengths of time. The images were investigated regarding variation due to the ageing length and due to the homogeneity of the connective tissue regions. The presented algorithm consists of the following main elements: (1) pre-processing of the spectra to overcome large quality differences in FTIR spectra and differences due to scatter effects, (2) identification of connective tissue regions in every image, (3) labelling of every connective tissue spectrum with respect to its location in the connective tissue region, and (4) analysis of variations in the FTIR microscopic images in regard to ageing time and pixel position of the spectra in the connective tissue region. Important spectral parameters characterising collagen and proteoglycan structure were determined.
Histological characterization of spinal fusions in Atlantic salmon (Salmo salar) has demonstrated shape alterations of vertebral body endplates, a reduced intervertebral space, and replacement of intervertebral cells by ectopic bone. However, the significance of the notochord during the fusion process has not been addressed. We have therefore investigated structural and cellular events in the notochord during the development of vertebral fusions. In order to induce vertebral fusions, Atlantic salmon were exposed to elevated temperatures from fertilization until they attained a size of 15g. Based on results from radiography, intermediate and terminal stages of the fusion process were investigated by immunohistochemistry and real-time quantitative polymerase chain reaction. Examination of structural extracellular matrix proteins such as Perlecan, Aggrecan, Elastin, and Laminin revealed reduced activity and reorganization at early stages in the pathology. Staining for elastic fibers visualized a thinner elastic membrane surrounding the notochord of developing fusions, and immunohistochemistry for Perlecan showed that the notochordal sheath was stretched during fusion. These findings in the outer notochord correlated with the loss of Aggrecan- and Substance-P-positive signals and the further loss of vacuoles from the chordocytes in the central notochord. At more progressed stages of fusion, chordocytes condensed, and the expression of Aggrecan and Substance P reappeared. The hyperdense regions seem to be of importance for the formation of notochordal tissue into bone. Thus, the remodeling of notochord integrity by reduced elasticity, structural alterations, and cellular changes is probably involved in the development of vertebral fusions.
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