# Critical Review of Techniques for Food Emulsion Characterization
## 3 Techniques for Emulsion Characterization  
### Microscopic Analysis  
One of the most straightforward and widespread methods for emulsion characterization is microscopic analysis. Microscopic analysis is performed to assess the morphology of the droplets (shape and size), droplet concentration, and distribution throughout the sample [66]. Observing changes in the morphology of the droplets over time can give us valuable information on emulsion stability and the mechanism of phase separation. The image typical for stable emulsion shows small, non-flocculated, homogenously distributed droplets that are relatively uniform in size (Figure 2a). Unstable emulsion separating through flocculation can be recognized by the presence of equally sized droplets that group but maintain their integrity. In the emulsions undergoing separation through coalescence or Ostwald ripening (Figures 2b and 3), small and large droplets are present at the same time [37]. Microscopic evaluation of emulsions can be performed via several techniques. The most common methods include optical microscopy, fluorescent microscopy, and electron microscopy [67; 68]. Optical microscopy is commonly used for the initial assessment of emulsions due to its simplicity, availability, and capability to quickly provide qualitative  
Figure 2: Optical microscopy images of (**a**) a stable emulsion, and (**b**) an unstable emulsion separating through coalescence. Adapted with permission from [66]. Copyright © 2023 Springer Nature. information about the emulsion. However, with the level of resolution it provides (down to 1-2 \(\upmu\)m [69], it is often insufficient for a detailed analysis of the emulsion structure. Optical microscope images of emulsions often suffer from relatively low contrast between the phases, caused by their similar refractive indexes, which makes it difficult to distinguish one phase from the other [70]. To improve the contrast and visibility of the droplets against the background of the continuous phase, water- or oil-soluble dyes absorbing light in the visible region can be added to the sample [66,71] or emulsion droplets may be stabilized with pigment particles (e.g., Irgalite Red D 3707, Cromophtal Violet D 5700, Paliotol Yellow K 0961) serving simultaneously as a contrast-enhancing agent and a surfactant (Figure 4) [72]. However, the color compounds added to the emulsions have to be carefully selected, as they may induce substantial changes in emulsion structure or interact with some of its components. Enhanced contrast can also be achieved using phase contrast or differential interference contrast microscopy, in which special lenses amplify the small differences in the refractive index into more significant differences in light intensity. Figure 4: Optical microscope images of Pickering emulsions stabilized with pigment particles: (**a**) Irgalite Red D 3707, (**b**) Cromophtal K 2960, (**c**) Paliotol Yellow K 0961, (**d**) Heliogen Grun K 8730, (**e**) Heliogen Blau K 6907, (**f**) Cromophtal Violet D 5700. Emulsions depicted in images (**a**,**c**–**f**) are W/O. The emulsion shown in (**b**) is O/W. Adapted from [73], published under a Creative Commons Attribution 3.0 License. Copyright © 2017 Royal Society of Chemistry. Figure 3: Optical microscopy image of coalescing emulsion droplets. Two droplets merge and form a larger one. Another limitation of conventional bright field light microscopy is limited imaging capabilities in the case of 3D objects, such as crystals of fat or ice (in frozen emulsions), or air bubbles. Polarization light microscopy is a valuable tool for three-dimensional imaging of such objects. As can be seen in Figure 5[74], there is a striking difference between images of fat crystals in emulsions observed using a transmitted light microscope and a polarized light microscope. Although conventional transmitted light microscopy allows for the visualization of emulsion droplets, it fails to clearly recognize transparent fat crystals against a bright background.