EXAMINE TISSUE COMPOSITION W/ ATTENUATED TOTAL REFLECTION IR MICROSPECTROSCOPY

保存骨组织样本的标准方法是将 石蜡或塑料(甲基丙烯酸甲酯/邻苯二甲酸二丁酯)。 然后，可以将它们以5-10米的厚度分段传输 红外线实验。这些薄片是必需的，因为 由磷酸盐和蛋白质提供的极大信号 对红外光谱的贡献。尽管嵌入提供了一个 这是检查骨骼矿物质成分的绝佳手段 该技术有几个缺点，包括(1)干扰 包埋介质的红外吸光度，以及(2)不确定 包埋过程是否影响蛋白质的二级结构。 可以使用传统的冷冻/切片切割方法来提供 原生组织样本。然而，要想做到更少就很难了 大于20微米，这是有问题的，因为高红外吸收 在这样的厚度下。因此，我们使用衰减全反射。 红外显微光谱学检查这类本地样品(例如。 不含塑料)。切片的组织被冷冻，并用 并用衰减全反射(ATR)进行检查。ATR 是一种表面技术，它大致探测到组织结构 10？m进入表面，这是基于 红外线辐射。由于这一特性，样本被检查 湿透了。ATR红外显微光谱分析是一种较好的分析方法 检查蛋白质光谱，因为没有变性条件可能 扰乱蛋白质结构是必需的。

A standard method for preserving bone tissue samples is to embed them in paraffin or plastic (methyl methacrylate/dibutyl phthalate). Then they can be sectioned at 5-10 ?m thicknesses for transmission infrared experiments. These thin sections are required due to the extremely large signals provided by the phosphate and protein contributions to the infrared spectra. Although embedding provides an excellent means for examining the mineral components of bone, this technique has a few drawbacks including (1) interference of the infrared absorbance of the embedding medium, and (2) it is uncertain whether the embedding process affects protein secondary structure. Conventional freeze/microtome cleavage methods can be used to provide native tissue samples. However, it is difficult to section to less than 20 ?m, which is problematic due to the high infrared absorbance at those thicknesses. Thus, we are using attenuated total reflection infrared microspectroscopy to examine such native samples (eg. without plastic). Sections of tissue are frozen and cleaved with a microtome and examined using Attenuated Total Reflectance (ATR). ATR is a surface technique that probes the tissue structure approximately 10 ?m into the surface based on the evanescent wavefront of the infrared radiation. Due to this property, the samples are examined wet. ATR infrared microspectroscopy is a superior method for examining protein spectra, since no denaturing conditions that may perturb the protein structure are required.