Boyde Alan
DPSU Queen Mary University of London, Mile End Campus, London, UK.
Methods Mol Biol. 2025;2885:621-670. doi: 10.1007/978-1-0716-4306-8_31.
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.
本章讨论了用于扫描电子显微镜（SEM）的骨和骨细胞样本制备方法。背散射电子（BSE）成像在骨领域是迄今为止最有用的，其次是二次电子（SE）和能量色散X射线（EDX）分析模式。样本可能具有三维表面细节，或者是无表面形貌的、经过抛光或微铣削的、树脂包埋块表面，或者是由骨基质包围的空间隔室的树脂铸型。在实验室之间运送湿骨样本最好用甘油。细胞的制备方法包括固定、干燥、观察骨细胞的底面以及进行金属导电涂层处理。详细描述了用碱性细菌蛋白酶、次氯酸盐、过氧化氢以及氢氧化钠或氢氧化钾进行浸软以去除细胞和未矿化基质的方法。特别关注骨样本的三维BSE-SEM成像方法。推荐使用聚甲基丙烯酸甲酯（PMMA）进行包埋，因为它易于微铣削或抛光。PMMA块的碘蒸气染色在BSE-SEM下能为细胞和未矿化基质提供出色的组织学图像。用PMMA或其他树脂包埋样本制作空间铸型是这种材料的一项重要用途。与其他成像手段的相关性很重要，这些手段包括共聚焦扫描光学显微镜、点投影X射线显微镜、显微放射ography和显微断层扫描。阴极发光（CL）模式的SEM成像可作为一种替代方法，用于观察诸如钙黄绿素和四环素等荧光矿化前沿标记。激光消融显微切割术允许将样本制备成用于与SEM结合的任何光学显微镜方法的载玻片。通过控制SEM样品室中的真空压力，可以消除“充电”问题，而无需施加表面导电涂层，从而使SEM使用起来快速简便。
