New method for quantitative polarised light microscopy of laser-ablation machined sections of bones and joints

Abstract

Bone Research Society meeting, Bristol, 25-27 June 2017, ‘Late breaking’ abstract Abstract ID: 106 LBP11 New method for quantitative polarised light microscopy of laser-ablation machined sections of bones and joints Alan Boyde Dental Physical Sciences, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London E1 4NS, UK Objectives: We have recently prepared very thin sections from the front face of bone blocks embedded in PMMA - which had previously been studied by backscattered electron scanning electron microscopy (BSE-SEM) and x-ray microtomography - by the new technique of laser-ablation machining. These sections justify the development and use of new 3D high resolution light microscopic methods. In conventional polarised light microscopy (PLM), positively birefringent crystals such as hydroxyapatite and/or negatively birefringent arrays of oriented molecules such as collagen appear brightest if they lie both in the plane of the section and at 45º to the axes of the crossed polarising filter elements. Birefringent elements appear black if they lie parallel to either polariser or analyser (or perpendicular to the plane of section). This situation prevents us from seeing the whole scene at once, because nothing can be seen in the dark sectors of the ‘Maltese cross’. Methods: We have overcome this problem by combining three grey-level PLM images. Digital images are recorded using green light with the polariser and analyser rotated 30º between each and used as red, green and blue components in a composite image. The colour maps the in-plane direction of the oriented molecular arrays irrespective of whether they are too small to be resolved. The intensity of the colour indicates the ‘strike’ of the molecules, i.e., the angle that they make to the plane of the section, brightest being parallel. An interpretive diagram has been developed which shows the colours for different orientations. This method has been applied to an array of large and small bone and joint samples. Results & Conclusion: Laser ablation microtomy produces high quality, thin sections of both hard, mineralised and dense fibrous connective tissues of any sort – even of single thin trabeculae – which can be studied with any light microscopic method as well as BSE-SEM. The novel 3x30º pseudocolour PLM images refine understanding of bone, cartilage, calcified cartilage, Sharpey fibre bone, ligament, calcified ligament, tendon, calcified tendon and fibrous periosteum structure in bones.