An improved method for the preservation and location of single cells during the embedding procedure for transmission electron microscopy

Single-cell specimen preparation for transmission electron microscopy has been technically difficult in some specific cells, such as in vitro fertilized eggs and microinjected zygotes of higher plants. A simple, highly efficient method is reported here which overcomes the difficulty by using a carefully designed protocol and fine handling devices. Leaf triangles of Salix matsudana L. are selected to mark the specimen during single cell embedding. The method facilitates easier sample handling and relocation of cells in embedded blocks. Furthermore, gradually increasing concentrations of glutaraldehyde and use of 9–13% mannitol as osmotium are shown to be suitable for zygote protoplast fixation. The method was successfully applied in the ultrastructural study of single zygotes, protoplasts of Nicotiana tabacum L., and microspores of Brassica napus L., indicating that the technique could be widely used in ultrastructural research of different kinds of single target cells of special interest.