Yeni Y N, Yerramshetty J, Akkus O, Pechey C, Les C M
Bone and Joint Center, Henry Ford Hospital, 2799 West Grand Boulevard, Detroit, MI, USA.
Calcif Tissue Int. 2006 Jun;78(6):363-71. doi: 10.1007/s00223-005-0301-7. Epub 2006 Jun 21.
Raman spectroscopy provides valuable information on the physicochemical properties of hard tissues. While the technique can analyze tissues in their native state, analysis of fixed, embedded, and sectioned specimens may be necessary on certain occasions. The information on the effects of fixatives and embedding media on Raman spectral properties is limited. We examined the effect of ethanol and glycerol as fixatives and a variety of embedding media (Araldite, Eponate, Technovit, glycol methacrylate, polymethyl methacrylate, and LR white) on Raman spectral properties (mineralization, crystallinity, and carbonation) measured from the cortical bone of mouse humeri. Humeri were fixed in ethanol or glycerol, followed by embedding in one of the media. Nonfixed, freeze-dried, and fixed but not embedded sections were also examined. Periosteal, endosteal, and midosteal regions of the intracortical envelope were analyzed. Raman spectra of fixative solutions and embedding media were also recorded separately in order to examine the specifics of overlap between spectra. We found significant effects of fixation, embedding, and anatomical location on Raman spectral properties. The interference of ethanol with tissue seemed to be relatively less pronounced than that of glycerol. However, there was no single combination of fixation and embedding that left Raman spectral parameters unaltered. We conclude that careful selection of a fixation and embedding combination should be made based on the parameter of interest and the type of tissue. It may be necessary to process multiple samples from the tissue, each using a combination appropriate for the Raman parameter in question.
拉曼光谱法可提供有关硬组织物理化学性质的宝贵信息。虽然该技术能够分析天然状态的组织，但在某些情况下可能需要对固定、包埋和切片后的标本进行分析。关于固定剂和包埋介质对拉曼光谱特性影响的信息有限。我们研究了乙醇和甘油作为固定剂以及多种包埋介质（环氧树脂、Eponate、Technovit、甲基丙烯酸乙二醇酯、聚甲基丙烯酸甲酯和LR白色树脂）对从小鼠肱骨皮质骨测得的拉曼光谱特性（矿化、结晶度和碳酸化）的影响。肱骨先固定于乙醇或甘油中，然后包埋于其中一种介质中。还对未固定、冻干以及固定但未包埋的切片进行了检查。分析了皮质骨内膜的骨膜、骨内膜和中间区域。还分别记录了固定剂溶液和包埋介质的拉曼光谱，以研究光谱重叠的具体情况。我们发现固定、包埋和解剖位置对拉曼光谱特性有显著影响。乙醇对组织的干扰似乎比甘油相对不那么明显。然而，没有一种固定和包埋的组合能使拉曼光谱参数保持不变。我们得出结论，应根据感兴趣的参数和组织类型仔细选择固定和包埋的组合。可能有必要对组织处理多个样本，每个样本使用适合所讨论拉曼参数的组合。
