电子显微镜的生物样品包埋前块染色技术探究

Exploration of en-block staining methods for preparing biological samples of electron microscopy

该研究在经典OTO技术基础上,探索比较5种可直接用于透射电镜(TEM)和扫描电镜(SEM)观察的包埋前块染色生物样品制备方法。通过优化试剂和实验条件,结果显示:四氧化锇、亚铁氰化物和硫代甲酰二肼等试剂的添加顺序及浓度对样品亚细胞结构有影响;OTO法虽有很强的图像对比度,但是不适用于果蝇眼睛这类较难固定渗透的组织;OFTO法(改自HUA的方法)在多种制样方法中成像效果最佳,可满足TEM及SEM双重成像要求;ROPA法制样过程中添加甲酰胺、连苯三酚可在无铅、铀等重金属染色情况下,得到较理想的对比度。

Based on the classical OTO technology,this study explores and compares five en-block staining methods for preparing biological samples that can be directly used for transmission electron microscopy(TEM)and scanning electron microscopy(SEM).By optimizing a variety of reagents and experimental conditions,the results show that the sequence of reagent treatment and concentration of osmium tetroxide,ferrocyanide and thiocarbohydrazide have an impact on the subcellular structure of the sample.Although OTO method has strong image contrast,it is not suitable for those tissues that are difficult to be fixed and penetrated such as Drosophila eyes.OFTO method(modification of HUA method)has the best imaging effect among various sample preparation methods,which meets the dual requirement of photographing under TEM and SEM.The addition of formamide and pyrogallol in the process of ROPA sample preparation can obtain ideal contrast under the condition of lead-free,uranium and other heavy metal staining.