Sample preparation of some plant tissues for transmission electron microscopy using high pressure freezing and freeze substitution

For sample preparation of biological block sectioning, high pressure freezing and freeze substitution (HPF-FS) is a better choice than chemical fixation. Freezing fixation can retain the original structures and preserve the soluble materials, and avoid the disadvantage of chemical fixation. Using high-pressure freezing, plant tissues are more difficult to freeze successfully than animal tissues and microbes because they have cell walls and varying degrees of vacuolation. For plant tissues, including the roots of Arabidopsis thaliana, 1-hexadecene is an ideal cryoprotectant/filler. The ability of highly vacuolated plant tissues (such as mature leaves and petals) to effectively remove intercellular air is crucial for successful high pressure freezing. Using the anthers of Arabidopsis thaliana, the leaves of Brachypodium distachyon, Axonopus compressus and Plantago asiatica, and the petals of Phalaenopsis bellina as examples, we found an effective method to remove air from the intercellular spaces: plant tissue in 1-hexadecene is placed in a vacuum desiccator, and then the air is removed using a vacuum pump, resulting in the release of numerous air bubbles. Plant tissue samples taken at different degassing times are then subjected to high pressure freezing and the subsequent required procedures of sample preparation. Based on the presentation of fine structures, the optimal time for different plant tissues can be determined and shows a positive correction with sample thickness. Good HPF-FS results show that the lipid bilayer within each organelle of the plant cells is clearly visible.