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Abstract Detail


Recent Topics Posters

Kitin, Peter [2], Lachenbruch, Barbara [1], Beeckman, Hans [2].

An inexpensive cryo-system for sliding microtome and light microscopy.

We developed a simple cryo-system which can be used on microscopes with epi-illumination, such as stereo, reflected light, fluorescence or confocal microscopes, and on the sliding microtome. Compared to commercially available stages, it is much less expensive and has higher flexibility with respect to sample configuration, but is not designed for high- precision regulation of a wide range of temperatures. This cryo-stage permits the use of short working distance objectives and observation of large samples. Because the specimen holder is custom-built it can be easily modified to accommodate samples of different sizes. The system utilizes a jet of cooled nitrogen gas to maintain the sample at -10°C to -70°C (depending on the flow rate). Water condensation on the surface of samples was eliminated by partial sublimation of the ice and clearing of the specimen surface with a jet of dry nitrogen gas, or the use of a specially designed cover slip covered with ethanol (melting point -112° C) as an immersion medium for the objectives. We currently use the cryo-system to investigate the occurrence of embolisms in xylem as well as development of bark in woody plants. We were able to fix wood and bark tissues in liquid nitrogen, cool them to -8 °C in a freezer, cut them at -30 °C on the sliding microtome, and then observe them frozen with a stereo or fluorescence microscope. Besides facilitating observation of native embolism, the system preserved cell shapes and cell contents without the artifacts that can result from drying and mounting samples. Fluorescence signals are stronger with less bleaching at these temperatures, and the images can be in color (rather than gray-scale from cry-scanning EM), so colors generated by labels, reagents, and stains can be observed.


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1 - Dept. of Wood Science and Engineering, Oregon State University, Corvallis, OR, 97331, USA
2 - Royal Museum for Central Africa, Laboratory for Wood Biology and Xylarium, Leuvensesteenweg 13, Tervuren, 3080, Belgium

Keywords:
fluorescence
cold stage
cryo-light microscopy
cryo-microtome
plant structure
Water relations.

Presentation Type: Recent Topics Poster
Session: P
Location: Ball Room & Party Room/SUB
Date: Monday, July 28th, 2008
Time: 12:30 PM
Number: PRT006
Abstract ID:1112


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