UEDA Katsumi Nara Women's Univ., Fac. of Sci., Prof., 理学部, 教授 (00031641)
KUROIWA Tsuneyoshi The Univ. of Tokyo, Fac. of Sci., Prof., 理学部, 教授 (50033353)
NOGUCHI Tetsuko Nara Women's Univ., Fac. of Sci., Ass. Prof., 理学部, 助教授 (00135823)
OSUMI Masako Japan Women's Univ., Dept. of Biol., Prof., 家政学部, 教授 (60060646)
TANAKA Kenji Nagoya Univ., School of Med., Prof., 医学部, 教授 (70013315)
Rapid freeze fixation has been recognized to provide preservation of ultrastucture of animal cells and bacteria, which is superior to that obtainable by conventional chemical fixation. This research project was planned to achieve rapid freezing of plant cells, which have been more difficult to freeze rapidly,probably due to their thick cell wall and watery vacuoles, and to promote understanding of structure of plant cell and dynamics of organelles during cellular process.
Submicroscopic architecture of plant cells and organelles was studied by rapid freeze-fixation used in conjunction with subsequent processing such as freeze-substitution, freeze francture and deep-etching freeze replica, and with transmission and scanning electron microscopy and fluorescence microscopy. Unicellular organisms, including yeast, microalgae, pollens protoplasts were rapidly frozen by dipping them into either liquid propane (ca. -190 C) or liquid freon (ca. -160 C) (Tanaka, Hirata, Osumi, Noguchi, Osafune,
Ueda). Metal contact method (slamming method) using liquid nitrogen (ca. -196 C) (Murakami, Osafune) and liquid helium (ca. -269 C) (Kuroiwa) was also adopted for rapid freezing of unicellular algal cells, protoplasts and isolated chloroplasts.
It has been demonstrated that rapid freeze-substitution fixation, when performed under the condition proper to the specimens, is a potent means with which watery palnt cells are fixed almost instantly while avoiding the artifacts due to ice crystal formation, and provides more realistic images of cellular ultrastructure. Superior structural preservation was most apparent in hydrophilic proteineous structures , such as cytoskeleton (Tanaka, Hirata, Osume, Ueda), filasomes (Tanaka, Osumi), plastid-dividing-ring (Kuroiwa) and H^+-atpase and phycobilisomes on photosynthetic thylakoid membranes (Murakami). Deep-etching freeze replica unveiled structure not only of intramembrane particles but also of phycobilisoms on the thylakoids (Murakami). This enables us to characterize their submicroscopic architecture and functional properties. Because of extremely short fixation time of rapid freezing, behavior of cytoskeleton (Tanaka, Hirata, Osumi, Ueda), filasomes (Tanaka, Osumi), mitochondria (Tanaka, Osafune), Golgi apparatus (Noguchi) and chloroplasts (Osafune, Kuroiwa) during various cellular processes, and the process of regeneration of cell wall in yest protoplasts (Osumi) were able to follow more accurately.
Rapid freeze fixation was shown to be useful for fluoro-cytochemical detection and immuno-gold localization of cellular components at submicroscopic level (Ueda). Less