## 2 Results and Discussion
### Self-Aggregation of FYNF-NH\({}_{2}\) Investigations
#### 2.4.4 Polarized Optical Light Microscopy (POM)  
A major factor determining the physical and chemical properties of self-assembled peptides (and amino acids) is molecular orientation [21]. Polarization-based optical analysis is very useful in molecular analysis, mainly when it comes to molecular orientation. Thus, in order to have a more in-depth perspective on the morphology of the peptide after incubation, polarized light microscopy was used. Accordingly, in Figure 6, the images obtained at two different polarizing angles (0\({}^{\circ}\) and 90\({}^{\circ}\)) and also at two different magnifications are represented. One may see that the self-assembled FEYNF peptide reveals intricate hyperbranched architecture, characterized by expansive dendritic patterns. Figure 6c highlights the increased visibility of short branches that have formed and are oriented nearly perpendicular to the nanofiber axis. This behavior was reported by [PERSON] et al., revealing the presence of entangled fibers following gelation, a characteristic of supramolecular hydrogels [17]. The FEYNF morphology can be mainly attributed to the presence of a tyrosine amino acid residue. It is well known that within physiological parameters (pH 7.4), the tyrosine residue likely retains its neutral state, facilitating pivotal interactions for the initiation of fibrillogenesis [47]. When juxtaposed with the previously studied peptide (FESNF-NH\({}_{2}\)) under analogous pH conditions [23], the introduction of tyrosine led to morphological changes for FEYNF-NH\({}_{2}\). Hence, although the formation of fibrils was absolutely absent in the case of the initial FESNF-NH\({}_{2}\) peptide at pH 7.4, and the newly synthetized FEYNF-NH\({}_{2}\) peptide possesses a distinctive capability at the physiological pH: the formation of dendritic-like fibrils (Figure 6). This structural variation predominantly stems from the tyrosine's aromatic ring and the presence of an adjacent hydroxyl moiety. Thus, in addition to the Phe-Phe interaction (previously shown to ensure \(\pi\)-\(\pi\) stacking and hydrophobic interactions), tyrosine may similarly contribute to peptide self-assembly. Interestingly, the theoretical calculated isoelectric point of the FEYNF peptide (6.94) closely aligns with the native FESNF peptide isoelectric point (6.99). Nonetheless, FEYNF-NH\({}_{2}\) exhibits resemblances in fibril formation with the amyloid-beta peptide (A\(\beta\)), especially when contextualized within the vicinity of its isoelectric point [48]. This noteworthy characteristic not only holds potential for designing related compounds but also offers fresh insights into the role of amino acids in triggering the emergence of gel aggregates. An interesting aspect is visible in Figure 6b,d registered at a polarization angle of 90\({}^{\circ}\). Under polarized light, the self-assembled FEYNF peptide exhibits birefringence. This indicates the self-assembly governed by the presence of peptide \(\beta\)-sheets, which led to well-organized, crystalline structures. It has been shown by others that temperature can also influence peptide self-assembly, resulting in bundles of aligned peptide nanofibers. These bundles can produce macroscopic birefringent domains on the order of tenths of millimeters [21; 49]. To further investigate using polarized light microscopy, we evaluated the Congo red staining assay. Congo red is acknowledged for its affinity to bind with \(\beta\)-sheets and adhere to amyloid structures, characterized by either complete or partial composition of \(\beta\)-folded sheets. In the context of this study, [PERSON] et al. observed that amyloid peptides tend to exhibit a distinctive \"apple-green\" birefringence upon Congo red staining when examined under polarized light [50].