Zhang Yan-Ru, Qiao Hai-Li, Ren Li-Li, Wang Rong, Lu Peng-Fei
The Key Laboratory for Silviculture and Conservation of the Ministry of Education, School of Forestry, Beijing Forestry University; School of Forestry, Inner Mongolia Agricultural University.
Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College.
J Vis Exp. 2020 Feb 3(156). doi: 10.3791/59251.
This report described sample preparation methods that scanning and transmission electron microscope observations, demonstrated by preparing appendages of the woodboring beetle, Chlorophorus caragana Xie & Wang (2012), for both types of electron microscopy. The scanning electron microscopy (SEM) sample preparation protocol was based on sample chemical fixation, dehydration in a series of ethanol baths, drying, and sputter-coating. By adding Tween 20 (Polyoxyethylene sorbitan laurate) to the fixative and the wash solution, the insect body surface of woodboring beetle was washed more cleanly in SEM. This study's transmission electron microscopy (TEM) sample preparation involved a series of steps including fixation, ethanol dehydration, embedding in resin, positioning using fluorescence microscopy, sectioning, and staining. Fixative with Tween 20 enabled penetrate the insect body wall of woodboring beetle more easily than it would had been without Tween 20, and subsequently better fixed tissues and organs in the body, thus yielded clear transmission electron microscope observations of insect sensilla ultrastructures. The next step of this preparation was determining the positions of insect sensilla in the sample embedded in the resin block by using fluorescence microscopy to increase the precision of target sensilla positioning. This improved slicing accuracy.
本报告描述了样本制备方法，通过制备蛀木甲虫（Chlorophorus caragana Xie & Wang，2012）的附肢，展示了用于扫描电子显微镜和透射电子显微镜观察的样本制备方法。扫描电子显微镜（SEM）样本制备方案基于样本化学固定、在一系列乙醇浴中脱水、干燥和溅射镀膜。通过在固定剂和洗涤溶液中添加吐温20（聚氧乙烯山梨糖醇酐月桂酸酯），在扫描电子显微镜下能更干净地清洗蛀木甲虫的虫体表面。本研究的透射电子显微镜（TEM）样本制备包括一系列步骤，如固定、乙醇脱水、树脂包埋、使用荧光显微镜定位、切片和染色。含有吐温20的固定剂比不含吐温20时更容易穿透蛀木甲虫的虫体壁，随后能更好地固定体内的组织和器官，从而获得清晰的昆虫感器超微结构的透射电子显微镜观察结果。该制备的下一步是通过使用荧光显微镜确定树脂块中包埋样本内昆虫感器的位置，以提高目标感器定位的精度。这提高了切片的准确性。
