We used polarized Raman spectroscopy to gain additional structural insights on amylin 20-29 and Aβ 25-35 fibrils. Raman anisotropy measurements on aligned samples can be employed to determine the relative orientation of chemical bonds and functional groups in fibrils, as first demonstrated by Lednev and coworkers. We prepared anisotropic samples of aligned fibrils for each polymorph using a drop coat deposition method. This method creates a "coffee ring" peptide film that forms because the evaporation of the solvent produces shear forces that move fibrils in the center of the droplet towards the perimeter. As the fibrils approach the perimeter of the droplet, they align themselves parallel to its edge with a high degree of ordering. We aligned the incident laser light to the edge of the coffee ring for the polarized Raman measurements. We designated a laboratory coordinate system (XYZ) where the Z-direction corresponds to the long axis of the fibrils (tangent to the coffee ring edge) and the Y-axis to the propagation direction of the incident laser light (Figure inset). Given the uniaxial symmetry of the fibrils, we only needed to acquire Raman spectra using four different combinations of incident and scattered light polarization configurations to obtain orientation information of chemical functional groups: ZZ, ZX, XZ, and XX (where the first and second letter indicate the direction of the incident and scattered light, respectively, along the laboratory coordinate system). The polarized Raman spectra of the three different fibril polymorphs are shown in Figure . As expected, the crosspolarized spectra (ZX and XZ, shown in blue and purple, respectively) overlap almost perfectly, indicating that there is no photodamage of the samples, no artifacts introduced by the different configurations of the polarization optics used (see Experimental Methods for more details), and that no displacements/rotations occurred in the samples during the measurement. The ZZ (black) and XX (red) spectra show the largest intensity variations. The most striking difference occurs with the Amide I A(0, 0) band, which is most intense in the ZZ spectrum and very weak in the XX spectrum.