# Critical Review of Techniques for Food Emulsion Characterization
## 3 Techniques for Emulsion Characterization
### Microscopic Analysis
As a result of the interactions between electrons and the specimen, electron signals (backscattered and secondary electrons), and X-rays are generated. Backscattered electrons are high-energy electrons scattered out of the sample that originate from deeper regions of the specimen. They provide valuable information about the composition of the sample. Secondary electrons are low-energy electrons, which have penetrated the surface regions of the sample a few nanometers below its surface. They reveal topographic information about the structure of the specimen surface. X-rays produced when electrons reach the sample give information about its elemental composition. These signals are collected by electron detectors to form a gray-scale image. The advantages of SEM that have made it one of the most popular tools for the ultrastructural analysis of emulsions include detailed topographical information of the sample surface, which cannot be obtained in two-dimensional projections produced by TEM, and a high depth of focus. The depth of focus at low magnifications can reach a few millimeters. However, preserving the shape and size of emulsion droplets during SEM  
Figure 6: A Pickering O/W emulsion (paraffin in water) stabilized with highly fluorescent carbon nitride quantum dots combined with solid laponite nanoparticles Adapted with permission from [78]. Copyright © 2018 Elsevier B.V.  
analysis is challenging, and requires specific SEM fixation protocols [81]. Other limitations include a lack of information on the internal structure of the sample, limited resolution, and the risk of damaging the specimen structure, especially after prolonged exposure to the electron beam [80]. TEM is one of the most powerful methods for the analysis of emulsion structure. The high resolution of TEM has rendered this technique an indispensable tool for imaging emulsions comprising droplets in the nanometer and sub-nanometer size range (down to 0.1 nm) [82,83]. Before TEM analysis, a 2-5 \(\upmu\)L sample of emulsion is deposited onto a carbon- or polymer-coated grid and dried for up to several hours. Alternatively, the sample may be cooled to cryogenic temperatures, which allows preservation of its native state. During imaging, a beam of electrons passes through an ultra-thin (<200 nm) sample of emulsion at high (60-200 kV) accelerating voltage. The electrons transmitted through the specimen are cast onto a fluorescent screen producing a high-resolution image. Typically, a bright field TEM imaging combined with diffraction mode is used for the characterization of the size and shape of emulsion droplets [84]. The main limitation of both SEM and TEM techniques is that they require relatively complex preparation of the sample before observation, which often involves dilution, spreading, drying, or freezing and may alter the original emulsion structure [71]. As a result, obtained images are often not representative of the sample in a liquid state [85]. The degree to which the original structure of the emulsion is maintained is highly dependent on the technique applied to fix the sample [80]. For example, chemical fixation with glutaraldehyde is known to distort native emulsion structure by causing the shrinking of oil droplets and is therefore considered unsuitable for emulsions [86]. Among other methods, fixation by cryogenic freezing, in which sample characterization is carried out in a vitreous frozen-hydrated state, has been shown as the most reliable [87]. In this approach, the water contained in the sample is transformed from a liquid to an amorphous solid state by vitrification without the formation of ice crystals. Avoiding the formation of ice crystals within the water phase allows the prevention of structural damage to emulsion droplets [88]. Cryogenic freezing can be used to fix emulsions before observation with both SEM and TEM [87].