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Development of a laser optical cryomicroscope for the study of freeze-preservation of plant gene resources.

开发用于植物基因资源冷冻保存研究的激光光学冷冻显微镜。

基本信息

批准号：
60840020
负责人：
YOSHIDA Shizuo
金额：
$ 15.36万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1985
资助国家：
日本
起止时间：
1985 至 1987
项目状态：
已结题

项目摘要

The objective of this project is to develop a new type of cryomicroscope for the observation of detailed structure of cells under the frozen state. For the optical system, a He-Ne laser beam is equipped. The laser beam is introduced into a Nikon Diaphoto TMD optical microscope through an optical glass-fiber cable and illuminated cell specimens through a half mirror. The images of the irradiated cells are viewed through an optical eyepiece or a SIT video camera equipped with a image enhancer device. When the laser beam is introduced by the optical glass fiber cable, the cell images are severely disturbed due to the speckle patterns. To eliminate the speckle patterns, the optical cable is randomly vibrated at a constant cycle. The light microscope is also equipped with bright field, phase contrast, differential interference contrast optics, or fluorescein illustration system.Cooling and rewarming of cell specimens are effected by a "conduction heat transfer" stage consisting of a copper cold sink interfaced with a transparent electrical resistance heater on which specimens are placed. Vaporized liquid nitrogen flows to the cold stage through a gas-flow controller system. the specimen temperature is measured with a 100 um copper-constantan thermocouple positioned at the center of the view region. Cooling and rewarming of cell specimens are achieved by introducing a small current generated from programmable voltage generator. The temperature accuracy during cooling and rewarming is in the range of +0.1゜C.By using this cryomicroscope, we can observe detailed structural changes of plant cells during freeze-thaw cycle. During freeze-thaw cycle of protoplasts, characteristic changes were observed on both plasma membranes and tonoplasts, suggesting a new information on the mechanism of freezing injury of plant cells.

该项目的目标是开发一种新型低温显微镜，用于观察冷冻状态下细胞的详细结构。对于光学系统，配备了He-Ne激光束。激光束通过光学玻璃纤维电缆引入Nikon Diaphoto TMD光学显微镜，并通过半透半反镜照射细胞标本。通过配备有图像增强器装置的光学目镜或SIT摄像机观察照射细胞的图像。当激光束通过玻璃光纤电缆引入时，由于散斑图案，细胞图像受到严重干扰。为了消除散斑图案，光缆以恒定的周期随机振动。光学显微镜还配备了亮场、相差、微分干涉对比光学系统或荧光素插图系统。细胞样品的冷却和复温受“传导热传递”阶段的影响，该阶段由铜冷却槽和透明电阻加热器组成，加热器上放置样品。汽化的液氮通过气流控制器系统流入冷段。用位于观察区域中心的100 μ m铜-康铜热电偶测量样品温度。通过引入由可编程电压发生器产生的小电流来实现细胞样品的冷却和复温。该低温显微镜在降温和复温过程中的温度精度在+0.1 ° C范围内。利用该低温显微镜，我们可以观察到植物细胞在冻融循环过程中的详细结构变化。在冻融循环过程中，原生质体的质膜和液泡膜都发生了特征性的变化，为植物细胞冻害机理提供了新的信息。