*10.4.1 Scanning electron microscopy (SEM)*

The protein morphology of the extracted collagen can be studied using SEM. The morphology of the extracted collagen can be compared to the standard bovine

#### **Figure 8.**

*SEM images of extracted collagen, with 1) acid-soluble collagen of Catla fish (a), pepsin-soluble collagen of Catla fish (B), acid-soluble collagen of Rohu fish (C) and pepsin-soluble collagen of Rohu fish (D) [139], (2) being from buffalo skin [14] and 3) being SEM image of porcine skin collagen [142] (used with permission).*

collagen available in the market. The expected microstructure of collagen from SEM images would be to observe collagen sheets which would be a combination of collagen fibrils and fibres that are bundled together to form a fibril network and dense sheet-like structure.

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Ramanathan et al. [140] used SEM to observe the surface morphologies of freeze-dried acid-solubilised fish skin collagen. The images showed a smooth surface texture, in two of the images, a layer-by-layer structure was observed (no definite fibres), and this was related to the intertwining of collagen fibres. Similarly, Rizk et al. [14], Tziveleka et al. [141], Rodrigues et al. [142], Pal et al. [139] all carried out SEM to assess the surface morphology of extracted collagen and all showed SEM images to have smooth or slightly wrinkled surfaces or sheet-like structures (**Figure 8**).

#### *10.4.2 Transmission electron microscopy (TEM)*

Transmission electron microscopy is usually carried out to observe collagen fibril structure and it is uniformity in a much deeper level. SEM only provides limited information on collagen morphology. **Figure 8** is showing an electron transmission image of mammalian lung tissue collagen at a magnification of 50 nm, while is showing a TEM image of collagen fibrils and fibres.

The preparative steps of collagen TEM are very specific and usually requires a technician to carry out each step carefully in order to observe the fibrillar structure of collagen. The Karnovsky fixative is mostly used as a preparative method prior to taking TEM images.
