## 7 Guidelines on Developing Biopolymeric Microparticles
### Characterization of Microparticles Loaded with Chemical and Biological Agents  
#### 7.3.1 Morphological Characteristics--Microscopical Observations  
After the suggested preliminary tests necessary to observe basic interactions between encapsulated material and material which will be used for encapsulation, and after the proper selection of the encapsulation procedure, characterization of microparticles may commence. Primarily, to observe the success of encapsulation, microscopic observations give valuable information. Not only about the size of microparticles, but the surface texture, proof of chitosan binding, the thickness of the membrane and can even reveal the presence of biological agents either on the surface or in the core. For example, basic optical microscopic observations can also reveal the percentage of shrinkage of microparticles after drying to the constant mass, as well as the collapse of the matrix and even loss of sphericity [133]. In the paper of [PERSON] et al. [132], a decrease in the size of the microcapsule can be observed with the increase in the Ca\({}^{2+}\) ions used in the encapsulation procedure. This is in correlation with other literature data, which revealed the formation of smaller particles with higher Ca\({}^{2+}\) concentration, due to the lower percentage of water retention and formation of tougher gel matrix [148]. Confocal laser scanning microscopy (CLSM) may reveal interesting data with regards to the observed microparticle. [PERSON] et al. [133] reveal data on chitosan binding over Cu-alginate microparticles. Microphotographs were taken under CLSM in fluorescence mode and transmitted light mode, which clearly shows the existence of the chitosan layer (stained with 0.01% Eosin Y, xanthene dye) on the surface of the microparticle. Eosin dye binds to the amino groups of chitosan and is, therefore, possible to observe the thickness of the coating layer. In this case, the thickness was about 7 \(\upmu\)m in the case of the microparticles loaded with only a chemical agent (Cu\({}^{2+}\)), and in the case of microparticles loaded with Cu\({}^{2+}\) and _T. viride_ spores, a layer was about 11 \(\upmu\)m thick. The different size in layer thickness over microparticles without _T. viride_ spores was explained as a lower extent of reaction  
Figure 5: Schematic representation of calcium-alginate microparticles preparation (microspheres and microcapsules) in two steps: (I) Ionic gelation (II) microsphere fabrication, and (III) polyelectrolyte complexation. between chitosan and alginate matrix. Electrostatic interactions between protonated amino groups on chitosan and ionized carboxylic acid groups on alginate are enhanced, due to the binding of _T. viride_ on the matrix. Similarly, in the paper of [PERSON] et al. [132], a clear existence of a thin chitosan layer on the surface of Ca-alginate microparticles was observed. Optical microscopy and CLSM may reveal data on microparticle size, general morphology, presence of biological agent (even protruding of hyphae from the microparticles) or chitosan layer, but scanning electron microscope (SEM) can shed light on even more details. In the paper of [PERSON] et al. [131], irregular wrinkles can be observed on dried microspheres as a consequence of biopolymer strain-relaxation processes, which are generally associated with water/humidity loss. Furthermore, SEM images can reveal pores and anomalies on the surfaces. For example, in the paper of [PERSON] et al. [131], a highly porous surface was observed (size of the pore is about 0.169 \(\upmu\)m), as well as numerous spherical blebs (_T. viride_ spores) which are visible as entangled into the matrix of a microsphere. Furthermore, this was confirmed in the publication of [PERSON] et al. [132], where the surface of microspheres without _T. viride_ spores is highly porous, and where numerous blebs are found on microspheres prepared with _T. viride_ spores.