To reveal finer structural details, we conducted a nanoscopic analysis by acquiring infrared spectra on single fibers imaged by AFM (see ESI). We exploited s-SNOM spectroscopy (scattering type scanning near-field optical microscopy) that is based on the coupling of AFM to cutting-edge infrared technologies to obtain information on nanoscale topography with chemical sensitivity. In particular, a typical s-SNOM spectrum is representative of the vibrational profile of sample surfaces that are 20 nm wide, with a penetration depth up to 100 nm.26, 27 Moreover, the interaction of the incoming radiation with the metallic tip of the AFM results in a strong polarization of the electromagnetic radiation along the direction perpendicular to the sample.28 Thus, s-SNOM peaks' intensities strongly depend on the orientation of covalent bonds. Figure shows the nano-IR average absorption measured on different fibers of the two-component assemblies. Figure shows the corresponding data acquired for the racemic gel composed of Hff and hFF and devoid of the Asp signal, as a control. First of all, all spectra measured on different fibers of the twocomponent samples of Fig. show the Asp signals, and in particular the distinctive signature below 1400 cm-1 that indeed is absent in the control without Asp (Fig. ). This data supports the hypothesis of co-assembled systems in both cases, as opposed to self-sorted ones, where two populations of fibers with distinctive profiles with and without Asp signals would be expected. Interestingly, despite showing similar frequency, profiles' spectra showed clear differences in peaks' intensities. Based on ATR results, which confirmed similar absorption profiles for the different samples (Fig. ), and given the polarized nature of the s-SNOM signal, this evidence hinted at differences in the three-dimensional arrangement of the assembled tripeptides that were likely to display a strong alignment of IR-active functional groups. To deepen this aspect, we performed a Principal Component Analysis (PCA) on single nano-IR absorbance spectra, whose results are shown in Figure4C-D. The first three components, PC1, PC2 and PC3, whose scores and loadings are shown respectively in Figure and, were taken into account, covering respectively 38%, 16% and 14% of the total data variance. The two-component systems have been here compared to the Dff assembly too (maroon markers). We first observed that Dff-Hff spectral distribution appeared to be more homogeneous compared to the other samples, as revealed by the area of the variance ellipses shown in Figure . This is likely related to a higher structural order of Dff-Hff in the assembly network compared to Dff-hFF, as expected, and surprisingly to Hff-hFF too. It is possible that the negative charge on Asp sidechain establishes ionic interactions with the cationic Nterminal groups, thus contributing to the organisation of the assemblies. Score plots revealed the clear separation of Dff-Hff from the other samples, toward positive values of PC1, of the Dff toward positive values of PC2, and of the racemic samples toward positive values of PC3. As shown in Figure , were PCA loadings are reported, PC1 shows a prominent peak around 1690-1700 cm-1 likely related to amide I modes29 that have been described for primary amides (i.e. terminal CONH2 groups),30 or t H-bound COO-groups.31 PC2 scores, whose loading show a main peak at 1645 cm-1, representative of amide I modes, group together the two-component systems in slightly negative values area, suggesting similarities in their amide 3D arrangements with damped amide I intensity compared to Dff, showing conversely positive PC2 values. Similarly to amyloid fibrils, the high alignment of amide bonds along the fiber axis produced indeed the reduction of the amide I band intensity.32 PC3 showed a main peak at 1550 cm-1, ascribed to amide II band and minor negative contributions of Asp COO-modes at 1586 and 1380 cm-1. It can be thus considered as a fingerprint of Asp, as also suggested by the grouping together of all Asp-containing samples.