Journal of Electron Microscopy Advance Access published online on June 16, 2006
Journal of Electron Microscopy, doi:10.1093/jmicro/dfl014
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Laboratory of Electron Microscopy/Open Research Center, Japan Women's University, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan; Non-Profit Organization: Integrated Imaging Research Support, Villa Royal Hirakawa, 1-7-5, Hirakawacho, Chiyoda-ku, Tokyo 102-0093, Japan
* To whom correspondence should be addressed. 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
Received January 10, 2006
Accepted April 28, 2006
Full-length paper
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
Masako Osumi 1 *,
Mami Konomi 2,
Tomoko Sugawara 3,
Tomoko Takagi 4,
and
Misuzu Baba 5
2 Laboratory of Electron Microscopy/Open Research Center, Japan Women's University, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan
3 Division of Material and Biological Sciences, Graduate School of Science, Japan Women's University, Japan; Basic Research Laboratory, Kanebo Ltd, 5-3-28, Kotobuki-cho, Odawara, Kanagawa 250-002, Japan
4 Laboratory of Electron Microscopy/Open Research Center, Japan Women's University, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan; 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
5 Laboratory of Electron Microscopy/Open Research Center, Japan Women's University, 2-8-1 Mejirodai, Bunkyo-ku, Tokyo 112-8681, Japan; National Institute of Natural Sciences, Nishigonaka 38, Myodaiji-cho, Okazaki, Aichi 444-8585, Japan
Masako Osumi, E-mail: osumi{at}fc.jwu.ac.jp
Abstract 
-1,3-glucan and its synthase during septum formation in Schizosaccharomyces pombe.-1; 3-glucan; ultra-low temperature LVSEM; immunoelectron microscopy; freeze-fracture replica labeling.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  J. Griffith and F. Reggiori Ultrastructural Analysis of Nanogold-labeled Endocytic Compartments of Yeast Saccharomyces cerevisiae Using a Cryosectioning Procedure J. Histochem. Cytochem., August 1, 2009; 57(8): 801 - 809. [Abstract] [Full Text] [PDF]
|
|