藓类植物扫描电镜观察材料的制备  

Sample Preparation for Scanning Electron Microscope in Moss

作　　者：刘永英[1,2] 权玉萍 李琳[2] 赵建成[2] 

机构地区：[1]焦作高等师范专科学校生物系,河南焦作454001 [2]河北师范大学生命科学学院,河北石家庄050024

出　　处：《实验室研究与探索》2016年第2期52-54,共3页Research and Exploration In Laboratory

基　　金：国家自然科学基金项目(31370237);河南省高等学校重点科研项目(15B180007);高等学校博士学科点专项科研基金项目(2013130312005)

摘　　要：分拣、清洗并剥离厚肋流苏藓(Crossidium crassinerve)叶片数十片,放入2.5%戊二醛溶液中固定3 h,经磷酸缓冲液漂洗后,使用梯度浓度酒精逐级脱水(50%、70%、80%、90%各5 min,100%共3次,分别为5、10和20 min),随后依次移入乙醇和乙酸异戊酯(1∶1)混合溶液以及乙酸异戊酯中分别置换15和20 min(或过夜),经临界点干燥2 h后将叶片(腹面朝上)贴到金属台上喷金镀膜,最后用扫描电子显微镜观察、拍照。对照实验是叶片经自然风干后直接装台观察。经该实验临界点干燥法处理的藓类材料细胞结构完整、形态饱满、表面光滑,丝体末端细胞的疣形态和数目清晰可辨,观察效果理想。未经临界点干燥处理的样品丝体细胞和疣极度皱缩、塌陷,无法辨别细胞界限,疣的形态和数目更是无从知晓。该方法适用于藓类各器官的扫描电镜观察材料制备。The aim of the work is to find out an optimizing method of sample preparation for scanning electron microscope（ SEM） in moss based on critical point drying. Dozens of Crossidium crassinerve leaves were peeled away from the sorted and washed plants,firstly these leaves were fixed in 2. 5% glutaraldehyde solution for 3 h,secondly they were dehydrated in increasing ethanol gradients,50%,70%,80% and 90%,each step for 5 min,finally in 100%with three times,for 5 min,10 min and 20 min,respectively. After being rinsed with phosphate buffer solution,thirdly they were displaced by ethanol and isoamyl acetate mixture solution（ 1∶ 1） and isoamyl acetate for 15 min and 20 min successively,or overnight,then by critical point drying for 2 h,they were stuck on the specimen stub and sputtered with a gold layer. Finally they were analyzed in an SEM. Untreated leaves were regarded as a control group. The filament cells of the moss have plump form,integrated structure and smooth surface,and the number and shape of papilla of filament end cells were very clear by the critical point drying. A control group of without the critical point drying showed that the filament cells shrank and collapsed seriously so that the boundary of the cells was invisible,and the shape and number of papillae were unclear. It can conclude that the critical point drying can avoid surface tension of moss cells,preserve the microstructure of the biological sample,and could be used to sample preparation for SEM for every organic of moss.

关 键 词：临界点干燥 藓类 扫描电镜 

分 类 号：Q94-3396[生物学—植物学] N33[自然科学总论]