Optimization of Specimen Preparation Methods for Cryo Electron Microscopy of Oil-in-water Emulsions

Emulsions are very important to several key industries such as water treatment (i.e. municipal/industrial wastewater), pharmaceutical, and petroleum [1, 2]. Crude oil is typically extracted from oil-wells by injecting water into the reservoir resulting in an extracted flow of water-oil-gas mixture. The water from the extraction process is referred to as produced water (PW). During the separation process both waterin-oil (W/O) and oil-in-water (O/W) emulsions are formed. The O/W emulsions are not readily removed during the oil and gas separation processes and remain in the PW, making discharge or reuse of the PW a challenge. Thus, a more efficient removal of the O/W emulsions is necessary to minimize their unwanted effects on the environment. However, the removal of oil from water demands a full understanding of oil and water mixing in these emulsions. In this regard, cryo scanning electron microscopy (cryoSEM) and cryo transmission electron microscopy (cryoTEM) can be utilized to obtain more detailed information about the emulsions with nanometer range resolutions [3]. There is still a need to address the effects of specimen preparation methods on the successful imaging of emulsions with these techniques. This study presents an investigation of sample preparation strategies for improved SEM and TEM analysis to characterize O/W emulsions in PW. Emulsions were synthesized by mixing ~1 % crude oil in water and then stabilizing them with sodium dodecyl benzene sulfonate (SDBS) surfactant. The specimens for cryoSEM were prepared by using plunge and high-pressure freezing (HPF) methods. Whereas the cryoTEM specimens were prepared with an automated plunge freezing system (VitrobotTM from FEI Company). SEM and TEM analyses were performed by utilizing Nova nanoSEM and Titan Krios instruments from FEI Company, respectively.