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Journal of Electron Microscopy Advance Access published online on March 16, 2009
Journal of Electron Microscopy, doi:10.1093/jmicro/dfp013
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© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Smart specimen preparation for freeze substitution and serial ultrathin sectioning of yeast cells

Masashi Yamaguchi1,*, Hitoshi Okada1,2 and Yuichi Namiki1

1 Medical Mycology Research Center, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8673
2 Non-Profit Organization, Integrated Imaging Research Support, Villa Royal Hirakawa 103, 1-7-5 Hirakawa-cho, Chiyada-ku, Tokyo 102-0093, Japan

* To whom correspondence should be addressed. E-mail: yama{at}faculty.chiba-u.jp

A smart and efficient method for freeze substitution and serial sectioning of yeast cells is described. Yeast cells were placed in a single layer between two copper disks, rapidly frozen, freeze substituted and embedded in an epoxy resin. The cell layer was re-embedded by the same resin, the surface trimmed leaving 1 µm above the cell layer, and serially sectioned. The sections were collected on the two-slit grids and placed on a Formvar film mounted to cover the holes of an aluminum supporting rack. The grids were removed from the rack, stained together using a silicon tube and observed in a transmission electron microscope. The images of yeast cells observed were clear and natural, and would be useful for a detailed 3D structural analysis such as structome.

Keywords     freeze substitution, serial thin sectioning, yeast, transmission electron microscopy

Received     16 September 2008, accepted 8 February 2009


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