Scanning Electron Microscopy and Bone.

This chapter discusses methods for preparing samples of bone and bone cells for scanning electron microscopy (SEM). Backscattered electron (BSE) imaging is by far the most useful in the bone field, followed by secondary electrons (SE) and the energy dispersive X-ray (EDX) analytical modes. Samples may have detail in a 3D surface, or be topography-free, polished or micromilled, resin-embedded block surfaces, or resin casts of space compartments surrounded by bone matrix. Shipping wet bone samples between labs is best done in glycerol. Methods for cells include fixation, drying, looking at undersides of bone cells, and metallic conductive coating. Maceration with alkaline bacterial pronase, hypochlorite, hydrogen peroxide, and sodium or potassium hydroxide to remove cells and unmineralized matrix is described in detail. Attention is given especially to methods for 3D BSE-SEM imaging of bone samples. PMMA is recommended for embedding because it is easily micromilled or polished. Iodine vapor staining of PMMA blocks gives excellent histology for cells and unmineralized matrix with BSE-SEM. Making spatial casts from PMMA or other resin embedded samples is an important use of this material. Correlation with other imaging means, including confocal scanning light microscopy, point projection X-ray microscopy, microradiography and microtomography, is important. Cathodoluminescence (CL) mode SEM imaging is an alternative for visualizing fluorescent mineralizing front labels such as calcein and tetracyclines. Laser ablation microtomy allows preparation of samples as slides for any method of light microscopy combined with SEM. Control of the vacuum pressure in the SEM sample chamber can be used to eliminate "charging" problems without the need to apply a surface conductive coating, making SEM quick and easy to use.