Correlative X-ray microtomography and light microscopy of hard tissue sections.

Abstract

There are large numbers of ground sections of calcified tissues in museum and anatomy department and private collections. Many of these light microscopy (LM) slides are rare, historically important, or impossible to reproduce, e.g., deriving from endangered or extinct species. We examined possibilities for retrospective study of such material with X-ray microtomography (XMT). Microscope slides are in an inconvenient form for XMT. To match the aspect ratio and scanning volume for XMT to the slides, and to economise on beam time, we scanned them in batches. For example, fifteen 75mm long by 25mm wide slides were taped together to make a 25mm square column which was rotated around its long axis when scanning: the coverslips and the slides providing the separation between the samples. We also fabricated special holders like transport containers to hold batches of five slides. We used the MuCAT2 scanner at QMUL, 90kV, to produce high contrast resolution, high dynamic range XMT. The spatial resolution was limited to 25µm due to the sample size. The central 25mm region of a stack took 24 hours to scan. Analysis used TomView, ImageJ and Drishti software. Slides were selected to include a range of skeletal and dental tissue types from human, elephant, dog, narwhal, black rhinoceros, prairie marmot and sperm whale. Accurate mineralisation determinations can be made from the XMT data except where sections are too thin and partial volume effects dominate the data. Section thicknesses need to be at least three voxels to allow accurate mineralisation determination - two outer voxels can be discarded, and the central voxel assumed good. Fortunately, most of the ground sections we examined were more than 100µm thick. We can, therefore, now report mineralisation densities for all the calcified tissues in the context of any mode of LM imaging. Here, we especially favour polarised light microscopy (PLM) since we have constructed an automated instrument with computer control. However, both quantitative and qualitative PLM require well cleared and plane parallel sections, requirements not always met in older material. Ethics statement: all samples studied antedated the UK Human Tissues Act 2004 and CITES regulations.