## 3 Selected Methods and Experimental Approaches for Analysis of HS Structure
### Solid State Investigations  
The structure of solid-state HS is less detailed than that present in the liquid state. However, various powerful methodologies such as solid-state NMR, electron microscopy and scattering of electromagnetic radiation, are available for researchers. Most of those investigations were analyzed in the framework of fractal theory applied to HS in solid state. 2.1 Microscopy Techniques: Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), Atomic Force Microscopy AFM  
Microscopy and imaging techniques have been extensively used to assess particle shape and aggregate structure of HS [81; 82]. The electron micrographs are able to highlight the fractal structure of microscopic irregular objects [83] whose fractal dimension d\({}_{f}\) (defined in the section Light/X-ray/Neutron Scattering Studies and Fractal Architecture of HS Aggregates), can be estimated by means of counting methods that make use of appropriate software for analyzing the digitized images. For example, a transition from collapsed structures with relatively smooth surfaces (at acidic pH) to open and more irregular structures with asymmetrical shapes (near neutral pH) was revealed from SEM microphotographs of soil HA particles, previously separated by aqueous suspensions at various pH values (3-7) after an equilibration time of 4 h [84; 85]. TEM image processing analysis performed on HA aggregates at different pH and equilibration times, confirmed the existence of fractal structures described by a power law between the mass and the characteristic length of the aggregates [86]. In particular, HA clusters exhibited large and compact structures at pH 3, which transformed into open and elongated structures at pH 6 at short equilibration times. However, with an increase in the equilibration time, larger and more compact structures were observed, as a result of the occurrence of aggregation process followed by internal restructuring of HA aggregates. Observing the structure of HS is challenging owing to their architectural complexity and dynamic nature. Additionally, these molecules can adopt multiple interchanging conformational states depending on the pH and metal complexation [57; 87]. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) have been employed for a long time to study the micro-morphologies of HA and FA [88; 89; 90]. Compared to electron microscopy, AFM offers more flexibility in sample preparation, three-dimensional imaging and, in many cases, better image resolution [91]. The advantage of AFM techniques is the ability to image microstructures not only on dried samples, but also in aqueous solutions, with a spatial resolution in the nanometer range [92]. AFM images can be obtained at ambient temperature and pressure, and the force can change during image acquisition. Thus, AFM is a valuable tool allowing to visualize small colloids, as well as colloid agglomeration, metals adsorption onto surfaces, or morphological changes affected by interaction with anions and pH [93]. However, imaging techniques require sophisticated sample preparation (e.g., mounting on slides or sample dewatering/drying), which is highly perturbing to the structure of the sample under study. Imaging also refers to the morphological analysis of the single particle which can be very laborious and can only visualize a sample that is not representative from a statistical point of view, even with the aid of automated image recognition systems. HA or FA were dispersed on mica as the better substrate not only because of its easily available atomically smooth surface but also its similarity in surface properties with the mineral materials in natural aquatic and terrestrial environments. Thus, AFM study results are very important for understanding the structure and function of HS in water and soil environments [94]. Indeed, HA, FA, and their respective metal complexes can form many different structures on mica surfaces as revealed by recent AFM studies [95].