李国平1,2 ，黄群策1*， 秦广雍1
(1.郑州大学离子束生物工程省重点实验室,中国河南 郑州 450052;
2.莆田学院环境与生命科学系,中国福建 莆田 351100）
摘 要：目的:为了更直观地观察和显示花粉和花粉管中细胞结构及其细胞核的状态与行为。方法:雪松花粉和花粉管经卡诺液固定，分别以埃氏苏木精、曙红、Hoechst 33243单染和曙红-Hoechst 33342双染后，用冬青油整体透明，在激光扫描共聚焦显微镜下观察。结果:4种染色法观察效果不同；以曙红-Hoechst 33342双染的样品观察效果最佳，在紫外光激发下清晰地显示出细胞核，在488 nm激光激发下不仅能清晰看到花粉和花粉管壁结构，且能分辨管细胞、柄细胞及体细胞的结构特点和空间位置关系。结论: 建立了一种快速简便的适于在激光扫描共聚焦显微镜下观察花粉和花粉管中成员细胞结构及其细胞核的状态、行为的制片技术；激光扫描共聚焦显微镜具有独特的共轭成像装置、连续光学扫描、图像三维重组和多通道检测等功能，极好地展示了雪松花粉和花粉管的结构特点，相比于传统的光学显微镜和荧光显微镜，其观察到的图像更清晰、更直观、更具立体感，是研究花粉生物学的理想工具。
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The Observation of Pollen and Pollen Tube of Cedrus deodara with Laser Scanning Confocal Microscope
LI Guo-pin1,2 , HUANG Qun-ce1*, QIN Guang-yong1
(1. Provincial Key Laboratory of Ion Beam Bio-engineering, Zhengzhou University, Zhengzhou 450052 ,Henan,China; 2. Department of Environment and Life science, Putian University, Putian 351100 , Fujian ,China)
Abstract: Aims:The aim of this study was to reveal the structure of pollen grain and demonstrated nuclei in the pollen and the artificially germinated pollen tube of Cedrus deodara. Methods: Following Carnoy fixation, the pollen and pollen tube samples were respectively treated with Ehrlich，s hematoxylin, eosin or Hoechst 33342 single-staining method and eosin/Hoechst33342 double-staining method, cleared in methyl salicylate and finally observed with Leica TCS SP2 laser scanning confocal microscope（LSCM）. Results: Different staining methods produced different results, but the eosin/Hoechst33342 double-staining method was believed to be the best. When the sample ,which was doubly stained with eosin and Hoechst33342, was excited by UV, the LSCM image showed three nuclei in one pollen grain clearly. When the sample was excited by 488nm laser, the LSCM image showed not only the pollen wall or pollen tube wall, but also the structure and spacial position of the tube cell, the body cell and the stalk cell in pollen grain or pollen tube. Conclusion: An ease and rapid procedure had been developed for the observation of pollen structure and the demonstration of nuclei in pollen and pollen tube with LSCM. Due to the critical features of confocal arrangement, serial optical scanning, advanced three-dimensional reconstruction and simultaneous multi-channel imaging, the LSCM could directly and visually display the three-dimensional architecture of the pollen and pollen tube with much more clarity than previously possible. The results of the present study suggested that the LSCM was an ideal tool to study the pollen biology.
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