Hisada A, Yoshida T, Kubota S, Nishizawa N K, Furuya M
Hitachi Advanced Research Laboratory, Hatoyama, Saitama, 350-0395 Japan.
Plant Cell Physiol. 2001 Sep;42(9):885-93. doi: 10.1093/pcp/pce132.
Metal-contact rapid freezing using liquid helium is theoretically the best method for preserving the fine structure of living cells with high temporal resolution in preparation of tissue samples for electron microscopy. However, this method is not commonly used, because of its technical difficulty and low reproducibility. We have designed and constructed an automatic device which allows simple, rapid and reproducible preparation of high-quality electron microscopic specimens by the non-specialist. We assessed the quality of cryofixation in samples prepared using this device by examining the preservation of cellular ultrastructure in relation to distance from the freezing block, and found that the region within 10 microm of the metal-contact plane was fixed with the highest quality. We applied this device, in combination with freeze-substitution methods and immunocytochemical techniques, to two phenomena involving rapid movement of subcellular components: (1) active movement of subcellular structures in the papillar cells of stigma and (2) light-induced rapid subcellular translocation of phytochrome A. Considering the importance of understanding subcellular processes of living cells for molecular and cell biology, this device will be a useful tool for diverse biological applications in the near future.
