Journal of Electron Microscopy Advance Access originally published online on June 16, 2006
Journal of Electron Microscopy 2006 55(2):75-88; doi:10.1093/jmicro/dfl014
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High-pressure freezing is a powerful tool for visualization of Schizosaccharomyces pombe cells: ultra-low temperature and low-voltage scanning electron microscopy and immunoelectron microscopy
1 Laboratory of Electron Microscopy/Open Research Center, Japan Women's University 2-8-1 Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan
2 Non-Profit Organization: Integrated Imaging Research Support, Villa Royal Hirakawa 1-7-5, Hirakawa-cho, Chiyoda-ku, Tokyo 102-0093, Japan
3 Division of Material and Biological Sciences, Graduate School of Science Japan Women's University, Japan
4 Basic Research Laboratory, Kanebo Ltd 5-3-28, Kotobuki-cho, Odawara, Kanagawa 250-002, Japan
5 Division of Biology, Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan
6 National Institute of Natural Sciences, Nishigonaka 38 Myodaiji-cho, Okazaki, Aichi 444-8585, Japan
*To whom correspondence should be addressed: E-mail: osumi{at}fc.jwu.ac.jp
Yeast cells have a thick cell wall composed of an inner network of glucans and an outer layer of mannoproteins, which is difficult to penetrate with osmium tetroxide. We previously developed the sandwich technique to overcome this problem. Although the freeze-etching method allows the fracturing of cryofixed yeast cells, it has been difficult to fracture cryofixed yeast cells for examination by cryo-scanning electron microscopy (SEM). The development of an alternative method of cryofixation, namely, high-pressure freezing, began in the 1960s and is now available for the electron microscopic analysis of yeast. We show here that when high-pressure freezing is combined with ultra-low temperature and low-voltage SEM using the new cryo-system, the Gatan Alto 2500 Cryo Transfer System, fractured and coated yeast samples could be quickly prepared. These samples yielded a fine fracture plane and revealed the ultrastructure of both external and internal cell components. We used this method to analyze the process of septum formation, one of the final and most important events of mitosis, and cell separation. The images we obtained provide a three-dimensional view of these processes for the first time. We also showed that high-pressure freezing in combination with immunoelectron microscopy made it possible to preserve the antigenicity, in situ localization, and behavior of the cell wall component 
-1,3-glucan and its synthase during septum formation in Schizosaccharomyces pombe.
Keywords High-pressure freezing, Schizosaccharomyces pombe, -1, 3-glucan, ultra-low temperature LVSEM, immunoelectron microscopy, freeze-fracture replica labeling
Received 10 January 2006, accepted 28 April 2006