水稻叶片透射电镜制样方法探讨

Transmission electron microscopic sample preparation method for rice leaf

水稻叶片表面有蜡质,其短细胞由硅质和栓质细胞组成。样品制备时包埋块有破洞。超薄切片常常出现锇黑颗粒,膜结构不清晰和图片反差小等现象。针对水稻叶片结构特点,本试验将戊二醛固定后的缓冲液漂洗次数增加到6次,漂洗时间约8 h,可充分漂洗叶片中的戊二醛,避免产生锇黑颗粒;用1%四氧化锇固定过夜,使膜结构清晰,图像反差好;树脂和丙酮不同比例(1∶3;1∶1;3∶1)渗透时间各12 h,纯树脂24 h,可使叶片被树脂充分渗透,包埋块无破洞,超薄切片完整。

The short cells of rice leaves are composed of silica and cork cells,with waxy surfaces. The sample block of transmission microscope was prepared with holes in it. In the ultra-thin section,osmium blacks are often present; the membrane structure is not clear and the image contrast is small. According to the characteristics of the rice leaf structure,rinsing the sample of experiment fixed with glutaraldehyde using buffer for 6 times and about 8 h can avoid the appearance of osmium blacks. Using 1% osmium for overnight fixation can make the membrane structure clear and image contrast good. The penetration with different ratios of resin to acetone（ 1∶ 3,1∶ 1,3∶ 1） for 12 h and then with pure resin for 24 h can make the leaf be fully penetrated by resin,and no hole presents in the block and the thin section is complete.