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

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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.