Ultra structure of oil-in-water emulsions a comparison of different microscopy- and preparation methods

We compare chemical fixation/ room temperature embedding in resin, cryofixation/ freeze substitution, and cryofixation/cryo imaging (freeze-fracture cryo-SEM) on several oil-in-water food emulsions. This is for visualization of the structure and thickness of the emulsifying layers consisting of food grade emulsifiers such as whey protein, sodium caseinate and milk phospholipids; layers that are expected to be in the range of only a few nm. Furthermore, the liquid nature and high water content of the samples further complicates the preparation process;especially since water is a major component of the samples. Concerning chemical fixation we adapted conventional protocols for preserving the emulsions by developing agar pockets for encapsulation or embedding in capillary tubes.Indeed, to use chemical fixation with these samples is challenging because we need to minimize alterations of the samples while ensuring at the same time that the samples are stabilized so they do not collapse when the water is removed.These protocols give an interesting view of the emulsions and the organisation of the interface layer surrounding the oil droplets. With cryofixation we could image more details of this interface and even the protein in the water phase. We observed that freeze substituted material seems to correspond very well to images of freeze fractured frozen samples in cryo-SEM where protein aggregates seems to be visible in the water phase.

With this work, we want to demonstrate the importance of combining different microscopic approaches to access the ultra structure of the oil-in-water emulsions due to their complexity and instability.