活体染色显微技术和绿色荧光蛋白在本科实验教学中的应用

The Application of Intravital Staining Microtechnic and Green Fluorescent Protein in the Experimental Course for Undergraduates

激光扫描共聚焦显微镜的原理和使用是本科生细胞生物学实验教学中的重要内容。目前,在细胞生物学实验教学中常使用绿色新鲜的植物叶片作为实验材料,在激光扫描共聚焦显微镜下对叶绿体的自发荧光进行观察。叶绿体的自发荧光信号强而且范围广,使学生难以清晰地理解特异性的荧光信号。该文通过对转p35S::Naa10-GFP基因拟南芥幼苗的根尖进行活体染色[碘化丙啶(propidium iodide,PI)和4′,6-二脒基-2-苯基吲哚(4′,6-diamidino-2-phenylindole,DAPI)染色],在不同的激发光下,收集相应的荧光信号,通过计算机辅助成像,获得不同颜色叠加的特异荧光信号图像。该实验设计简单可行,获得的图像清晰且便于观察,能够使初学者直观并且深刻理解激光扫描共聚焦显微镜的原理和使用方法,适合在高校细胞生物学实验教学中推广,同时也为研究其他蛋白质亚细胞定位提供技术参考。

The principle and usage of laser scanning confocal microscope are very important content in cell biology experimental course for undergraduates. In the current teaching procedures, green fresh leaves are used as material to observe the auto-fluorescent of chloroplast. The auto-fluorescent signal of chloroplast is strong and extensive, which leads to the result that the students cannot understand the specific fluorescent signal clearly. In this paper, the author stained the root tip of p35S：：Naa10-GFP transformed Arabidopsis with PI（propidium iodide） and DAPI（4′,6-diamidino-2-phenylindole） and collected different fluorescent signal under relative wave length laser. Specific fluorescent signals merged images were obtained by using computer aided imaging technique. The design of this experiment is simple and the resulted images are clear and easy to observe, which make the beginners understand the principle and usage of laser scanning confocal microscope easily and deeply. The method introducedin this paper can be generalized in undergraduate experimental course in colleges and provides technical reference to study subcellular localization of a protein.