**4.2 Morphology**

Different microscopy techniques can be used not only to observe the nanocapsule morphology and structure, but also to determine the average size, elemental composition, and state of aggregation. Scanning electron microscopy (SEM) or transmission electron icroscopy (TEM) are the most common techniques, and their choice depends on the size of the studied system and the established purposes [23, 45]. The principle of SEM is to scan the sample with a high-energy electron beam, and image formation is achieved by collecting low-energy secondary electrons or backscattered electrons that are released from the sample surface. For this reason, SEM images present a three-dimensional appearance and are useful to appreciate the structure, shape, and surface defects of the sample [45, 46].

Compared to SEM, TEM needs a higher voltage, resulting in higher resolution (0.2 nm). Since electrons can pass through the sample, the internal structure, whether crystalline or amorphous, can be observed [44]. Both techniques are expensive and require high vacuum, the main difference laying in the preparation of the sample and the information obtained from it. SEM requires sample conductivity, which is usually achieved by coating the sample with a thin layer of gold or platinum. In contrast, for TEM analysis the sample must be thin enough to be electron-transparent [45]. It is worth mentioning that a qualitative or semi-quantitative elemental chemical analysis can be performed by electron microscopy, coupling energy-dispersive X-ray spectroscopy (XEDS) [24]. In addition, the use of scanning transmission electron microscopy (STEM), a technique that combines both principles, has been reported for the characterization of micro- and nanocapsules [47].

Another useful tool for simultaneously determining particle shape, surface structure, and even some mechanical properties is atomic force microscopy (AFM). AFM images are obtained by measuring the displacement of the AFM tip during a raster scanning over the immobilized sample. The result is a high-resolution 3D profile of the surface under study. The principal advantage of AFM over electron microscopy is that it permits the imaging of almost any type of surface and biomolecules under different physicochemical conditions, during biological processes, or even the study of the mechanical properties of delivery systems at the nanoscale [45, 48].
