I preprared curcumin-loaded liposome by Thin Film Hydration Method using lecithin, curcumin and beta-sterol. I stained it with phosphotungstic acid and uranium acetate and took TEM images of each. TEM images showed that it was spherical but not show multilayered membrane structure. It's more like micelles. How can I adjust my fabrication method of liposome in order to correctly photographed TEM structures of liposomes? Should I increase the percentage of phospholipids?
The issue lies not in the fabrication of the liposomes but in the imaging technique itself. Water is an essential component of liposomes. Without water, lipids cannot self-assemble or maintain their conformation. When you place a sample on a grid, it inevitably dries out. Then, when you insert the sample into the TEM column, where a vacuum is maintained, the water evaporates almost instantaneously, leaving behind lipid debris, which you observed and identified as liposomes. While you can speculate that these aggregates were once liposomes, observing a multilayered structure requires maintaining water within them.
Cryogenic TEM is the only method suitable for such purposes. In cryo-TEM, the sample is vitrified, trapping water in an amorphous state inside the liposomes. This preserves the structure, allowing you to observe multiple layers.
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