To obtain a complete image of how a material is built up the structure has to be characterized at different levels. To be able to fully describe the structure it needs to be understood how the different parts of the system interact and all together build up the structure in three dimensions. Structural differences in a material can already be detected by the naked eye. Using a higher magnification, but still on an overall level of structure, we can see how small constituents have formed into e.g. a network structure, clusters and different phases etc. At a high resolution information can be obtained on how macromolecules associate into supramolecular assemblies, as molecules in a solution or small aggregates in dispersion. Supramolecular assemblies form strands in a network either as aligned molecules or as linked aggregates or particles. The packing of molecules, as well as the geometry and flexibility of strands, has a bearing on the network properties.
Using several different microscopy techniques it is possible to visualize different features at different structural levels and also dynamic course of events such as the mechanisms taking place in a product when it is produced, used, experienced or broken down. Examples of such mechanisms are aggregation, phase separation, crystallization, flocculation and dissolution which can occur during heating, freezing, melting, mixing, tension or storage. These mechanisms and the structures they give rise to have impact on many important physical and functional properties such as water-holding, diffusion and rheological properties.
Knowledge of the relationship between microstructure and functional properties of materials makes it possible to tailor-make structures for specific purposes within sectors such as foods, paper, paint, pharmaceuticals and hygiene.
Some microscopy techniques (LM, TEM) require that the material has to be very thin to get the light through the sample. If the material is a liquid a simple smearing of the sample is enough to visualize the structure using LM. To achieve higher resolution than what is possible in LM, i.e. higher than 0.5m, electron microscopy is required and thereby control of water. Two main alternative routes exist for the preparation of aqueous bulk samples for TEM; dehydration and solidification. When dehydration is used the water is replaced with a synthetic polymer in a process called embedding. When samples are embedded they are generally chemically fixed and dehydrated before. The embedded sample is sectioned and stained for different components in the sample.
When solidification with freezing is chosen, the most common way to analyze a fractured surface is by using “freeze-etching” which results in a replica of the fractured surface. The frozen sample itself can also be analyzed by using the plunge freezing technique. Freezing techniques are commonly used for multiphase systems containing fat, since dehydration steps can affect the fat phase and interfacial structures. High resolution metal shadowing, Mica, is suitable to use for examination of single biopolymers and supramolecular assemblies (diluted liquid samples). In this technique a monolayer of molecules is adsorbed on a mica surface and rotary shadowing performed at a very low angle.
It is necessary to have good knowledge of preparation techniques in order to avoid artefacts and understand how the preparation technique in itself affects the structure of complex material systems. When using freezing techniques it is important to have a fast freezing rate to avoid formation of ice crystals. Ice crystals are formed in the temperature interval between 0°C and 130°C, which means that the freezing media have to have a temperature below the ice crystal formation temperature and that samples have to be thin. Cryoprotectants, like glycerol, can also be added to reduce the temperature range in which ice crystals are formed. Visualizing of semi-solid and solid samples using LM require similar preparation as samples for TEM. The different phases in the materials are thereafter differentiated by specific staining or immunolabbeling to be seen in the microscope.
The CLSM technique allows examination of the build-up if bulk samples without any pre-preparation, except for staining, since the CLSM detects structures by using a laser source instead of transmitted light. Products and materials with a sensitive structure can therefore easily be analyzed with the CLSM technique because it is a nearly non-destructive technique. This is because the preparation needed has often limited effect on the specimen. Furthermore, it is also possible to analyze and determine changes in microstructures during dynamic course of events e.g. heating, freezing, tension and diffusion. To make the different structural components visible in the CLSM the sample is labelled with fluorescent staining specific for the component of interest. Certain materials are fluorescent in itself and thus do not have to be stained.