To determine the sub-tissue structural zonal boundaries in articular cartilage, a novel infrared (IR) microscopic imaging method based on the dichroic nature of the amide components has been developed and is discussed in this article. Thin canine cartilage-bone sections embedded in paraffin as well as in poly(methyl methyacrylate) (PMMA) were imaged under two orthogonal polarization states at 6.25 μm pixel size. The depth-dependent anisotropy of the amide components at perpendicular polarization states attributed by the collagen constituent in cartilage was analyzed. Since the transitional zone fibers are randomly arranged and the dichroic ratio value reaches unity in this zone, it is possible to identify the transitional zone boundaries, thus dividing the whole-depth tissue into three structural zones (superficial, transitional and radial). The zone division results from the infrared method agree well with the results from the established polarized light microscopy (PLM) method, which promises the potential of infrared imaging as an independent technique for the zonal boundary determination. The advantages of this dichroic ratio method are (1) it is independent of mode of operation (transmission/reflection), (2) it is independent of sample thickness, (3) either a polarizer or an analyzer can be used in experiments to determine zonal boundaries, and (4) it is sample orientation independent.

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