Abstract
The method of application of negative stains and the subsequent chemical interaction with various types of phospholipids affect vesicle morphology. Temperature, pH, stain concentration, and the method by which the vesicle solution is applied to the grid necessitates a systematic study. We have developed a method for spray mounting and uranyl acetate (UA) staining of extruded vesicles composed of dilauroylphosphatidylcholine (DLPC, 95%) and dimyristoylphosphatidylcholine (DMPC, 5%) from which we could acquire quality stereo images with high resolution field emission STEM.The vesicles were prepared by 19 passes through a low pressure extruder containing a 0.1 μm polycarbonate membrane filter. After extrusion, 0.5 ml of vesicle solution was diluted with 3.5 ml of 0.1 μm filtered water and vortexed. The concentration of diluted phospholipid was 1.35 mM. The vesicle solution and 2 % UA stain in Milli-Q purified deionized water were mixed at room temperature 1:1 v/v. The UA solution was first prepared at 2X the desired concentration (4%) and sonicated into aqueous solution for at least 15 min. Before mixing with the vesicle solution, the UA was diluted to the desired 2% concentration and run through a 0.1 ¼m filter.
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