**3. Results**

#### *3.1. Microscopic Imaging of the Films and Capsules*

At the first stage of the study, the prepared GA and GAC films were observed prior to and after submersion in HCl. Macroscopically it was visible that the samples after the acid treatment became opaque and swollen. Under the microscope, the untreated samples had a smooth surface with no structures visible [6]. As presented in Figure 3, the films after submersion in acid revealed a network-like structures, resembling scaffolds.

**Figure 3.** SEM image of GA and GAC films after 2 h in 0.1 M HCl. Scale bar: (**a**) 10 μm, (**b**) 1 μm.

There are clear differences between the images of a top and a middle layer of the sample (Figure 4). It appears that, after 2 h in acid, noticeably less solid material is left on the top of the film, than in the deeper part. The signals registered by CLSM can be potentially both from CAP and gelatin, due to very similar autofluorescence behavior. However, it is suspected that the outer layer consists mostly of CAP, while in the inner layer a swollen and undissolved gelatin can be present as well.

**Figure 4.** Confocal laser scanning microscopy (CLSM) images of GA film after 2 h immersion in 0.1M HCl: surface layer (**a**) and the inner central part (**b**) of the film.

Raman microscopy investigation was performed on GAC films before and after immersion in 0.1 M HCl. Several points have been scanned to obtain Raman spectra, which have been overlaid and compared. The spectra are shown in Figures 5 and 6.

As it can be seen from the spectra in Figure 5, the surface of the GAC sample is chemically uniform, without any phase separation visible. The acid-treated GAC samples display similar pattern in the spectrum as the untreated GAC. The spectra of the non-modified film (GEL) are not presented in the figure, but they were not different from the spectra of GAC. In Figure 6, different sets of spectra are overlaid. It appears that, in the untreated samples, the gelatin signals are overlapping with the peaks of CAP. After the acid-treatment, the signals from gelatin are weaker, but the signals from CAP are ye<sup>t</sup> undetectable. This outcome can be explained by presence of a small amount of gelatin-rich phase residue undissolved in acid and covering the CAP scaffold. This corresponds well with SEM and CLSM results described above.

**Figure 5.** The Raman spectra of several points examined on the surface of GAC film: (**a**) before immersion in acid; (**b**) after immersion for 2 h in 0.1M HCl. Multiple overlaid spectra are presented on each graph.

**Figure 6.** *Cont*.

**Figure 6.** Comparison of the Raman spectra: (**a**) untreated and acid-treated GAC; (**b**) acid-treated GAC and cellulose acetate phthalate (CAP) film (without gelatin). Multiple spectra of each composition are presented.
