Fine-tuning transmission electron microscopy methods to evaluate the cellular architecture of Ulvacean seaweeds...

Chemical fixation is a critical step in the analysis of the ultrastructure of seaweeds because the wrong approach can compromise the ability to distinguish fine-scale cellular composition. Fixation agents, fixation time and type of tissue are important factors to consider for transmission electron microscopy (TEM), and not every protocol is suitable for all cell types. We evaluated a range of fixation agents, post-fixation time and dehydration solutions to determine a TEM protocol for seaweeds in the Family Ulvaceae. We assessed Ulva lactuca using 5 protocols. The level of preservation obtained differed markedly between fixation methods The best result was obtained by fixing the sample with 2.5% glutaraldehyde, 0.05M sodium cacodylate buffer and 2% paraformaldehyde overnight, and 8h post-fixation in 1% in osmium tetroxide 1%. This approach and fixation time ensured that the membranes, especially the thylakoid membranes of chloroplasts, remained intact. Ethanol is recommended for dehydration as the use of acetone for dehydration resulted in the collapse of cellular membranes. This new protocol will ensure the ultrastructure of Ulvacean seaweeds can be clearly ascertained in the future.