Low-temperature embedding procedures applied to chloroplasts

Pea and spinach chloroplasts were fixed with glutaraldehyde and dehydrated with ethylene glycol at 0°C. In some experiments dehydration was achieved by the resin itself. The material was infiltrated at temperatures between −35 and 0°C with mixtures of hydroxypropylmethacrylate and butylmethacrylate or a mixture of methacrylates and acrylates containing crosslinking agents. Polymerization was achieved, at the same temperature as the infiltration, using uv light and benzoin methyl ether as the catalyst. In uranyl acetate stained sections of specimens embedded at −35°C the thylakoid membranes appeared light between the strongly and evenly stained partition gaps. Dark spots of various shapes appeared in the middle of each thylakoid intercalated at irregular intervals by closely apposed membrane segments. In specimens embedded at −18 or 0°C the dark spots were replaced by a continuous dark layer identical to the partition gaps. The patterns seen in sections of embedded chloroplasts also appeared in specimens prepared by cryoultramicrotomy. The structural patterns described deviate markedly from those obtained when a conventional procedure is applied to the material mentioned.