Stoffel M H, Frethem C, Hamilton D W, Friess A E
Institut für Tieranatomie, University of Bern Veterinary School, Switzerland.
Mol Reprod Dev. 1993 Feb;34(2):175-82. doi: 10.1002/mrd.1080340209.
Various fixation protocols were used in an attempt to improve preservation of rat epididymal sperm for high-resolution low-voltage scanning electron microscopy (HR-LVSEM). Wash solutions and fixatives of different composition and osmolarity were tested. Paraformaldehyde and glutaraldehyde concentrations were varied between 0.5% and 3%. Ruthenium red was tested as an additive in both primary fixation and postfixation, or in postfixation alone. HR-LVSEM revealed various degrees of ruffing, folding, blebbing, and peeling off of the plasma membrane, as well as holes of different sizes. The plasma membrane overlying the acrosome and the connecting piece proved to be particularly sensitive to varying fixation conditions. Consistent topographical differences were revealed among the different domains over the sperm head. Most of the differences were considered to be artifacts. Their consistency, however, suggests that structural and biochemical differences exist either within the membrane or in the structures subjacent to the membrane. Primary fixation turned out to be less critical than postfixation. Preservation of a smooth plasma membrane without holes could only be achieved when primary fixation in low aldehyde concentrations, with or without ruthenium red, was followed by postfixation with OSO4 and 1,000 ppm ruthenium red. Examination of thin sections of the same material confirmed that even a considerable number of small holes are difficult to detect in transmission electron microscopy. These results show that with the recent increase in resolution of LVSEM there is need for further effort to improve sample processing.
为了改善大鼠附睾精子的保存效果以用于高分辨率低电压扫描电子显微镜（HR-LVSEM）观察，采用了多种固定方案。对不同成分和渗透压的洗涤液及固定剂进行了测试。多聚甲醛和戊二醛的浓度在0.5%至3%之间变化。钌红作为添加剂在初次固定和后固定中进行了测试，或者仅在后固定中进行了测试。HR-LVSEM显示出质膜有不同程度的起皱、折叠、起泡和脱落，以及不同大小的孔洞。顶体和连接段上方的质膜对不同的固定条件特别敏感。在精子头部的不同区域发现了一致的地形差异。大多数差异被认为是人为假象。然而，它们的一致性表明膜内或膜下结构存在结构和生化差异。结果表明，初次固定不如后固定关键。只有在低醛浓度下进行初次固定（有无钌红均可），然后用四氧化锇和1000 ppm钌红进行后固定，才能获得无孔洞的光滑质膜。对相同材料薄片的检查证实，即使在透射电子显微镜下也很难检测到相当数量的小孔。这些结果表明，随着最近LVSEM分辨率的提高，需要进一步努力改进样品处理。
