# Critical Review of Techniques for Food Emulsion Characterization
## 3 Techniques for Emulsion Characterization
### Microscopic Analysis
The visibility of fat crystals under polarized light is greatly improved; however, this method fails to show the structure of the emulsion in the background. For this reason, it is advisable to combine several different microscopic techniques to obtain reliable information about emulsion structure. The techniques widely used to complement the information about emulsion structure gathered from white light microscopy are fluorescent microscopy and confocal laser scanning microscopy (CLSM) [75]. These approaches can be applied to assess emulsions containing compounds that display inherent fluorescence when exposed to light ( autofluorescence), which is typical for many oils (e.g., aromatic hydrocarbons), vitamins (e.g., A, B\({}_{2}\), E, and D), proteins, toxins (e.g., aflatoxin), metabolites, plant pigments (e.g., chlorophyll), and flavoring compounds [76]. Auto-fluorescent compounds can easily be differentiated from other emulsion components, as well as particles and air bubbles that may be present in the sample. The fluorescence of the oil phase can also be enhanced by adding fluorescent oleophilic dyes such as 3-alkoxyflavone or Nile Red [77]. Non-fluorescent compounds present in the emulsions, such as proteins, surfactants, contaminants, etc., can be visualized using fluorescent probes that selectively bind to target molecules. In addition, the fluorescence emitted by these molecules can be utilized for quantitative analysis of their content in the emulsion by fluorescence spectroscopy. Fluorescent dyes can be used to analyze the structure and stabilization mechanisms of emulsions. High-contrast images of emulsions comprising non-fluorescent oil and water phases can be obtained by labeling the oil-water interface using fluorescent stabilizers, such as carbon nitride quantum dots, combined with solid supports, e.g., laponite nanoparticles  
Figure 5: Fat crystals formed during the destabilization of canola oil-in-water (O/W) emulsion by freeze-thawing as a result of partial coalescence of canola oil droplets. Adapted with permission from [72]. Copyright © 2016 Elsevier Ltd.  
(Figure 6) [78] or fluorescent dyes, e.g., NBD [79]. Using fluorescent surfactants to stabilize emulsions can provide information on the localization and behavior of emulsifier molecules at the oil-water interface. [PERSON] et al. [78] have demonstrated that NBD, a common fluorescent dye, can effectively act as a surfactant. However, they have also revealed that the dye substantially affected the behavior of other particles adsorbed to the liquid-liquid interface, which altered the interfacial tension and the particle contact angle. This should be taken into account in studies utilizing fluorescent probes for labeling and _in situ_ imaging of emulsion components. Fluorescent microscopy and CLSM are typically employed for the visualization of emulsions containing droplets with sizes down to \(\sim\)300 nm [69]. Since, over the last few decades, the size of emulsion droplets has reached the lower end of the nanometer scale, visualization by optical microscopy has become insufficient to provide reliable data on nanoemulsion structure. Therefore, optical microscopy has been largely replaced by electron microscopy, which offers much higher resolution and allows imaging structures with sizes down to 0.1 nm [69]. The excellent resolution of electron microscopy allows visualization of multi-lamellar structures, vesicles, micelles, crystals, and liposomes, which remain undetected by both optical microscopy and particle size analyzers [80]. Electron microscopy techniques can be divided into transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The SEM analysis offers the advantage of obtaining topographical information at a significant depth of focus on a 2D image [80]. SEM generates images of a sample by scanning the surface with a focused beam of electrons accelerated under high voltage. The electrons penetrate the specimen and are deflected by elastic scattering.