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Second-harmonic and two-photon imaging and polarimetry of articular cartilage

Articular cartilage possesses an extensive extracellular matrix consisting of a highly organised network of collagen fibres embedded in a much finer mesh of proteoglycans and other glycoproteins. Many fundamental issues of cartilage biomechanics, its ageing and the development of osteoarthritis concern the detailed organisation of this matrix. Here we investigate the application of multi-photon microscopy to characterise the structure of the extracellular matrix. In reflection mode both second harmonic Generation (SHG) and two photon fluorescence (TPF) imaging modalities reveal differences in the pericellular and inter-territorial matrix in normal tissue and additional changes in degenerative lesions. The SHG signal from the surface zone is dependent on the direction of polarization of the laser excitation beam but the TPF signal is not. The former can be quantified to determine fibre orientation although the pattern is less well resolved than in tendon, reflecting the less regular orientation of the finer fibres. Nevertheless, previously unreported subtle variations in fibre orientation over the surface of the cartilage can be observed. In order to characterise variations with depth we carried out polarization sensitivity experiments at depths up to 180 microns into the tissue. At greater depths the polarization sensitivity is affected by the birefringence and dichroism of the overlying tissue and we have quantified these effects to allow correction of the data.