In Situ 3D Structural Determination of Soft Biomaterials by Computationally Assisted Polarized Raman Spectroscopy

Soft biomaterials, characterized by structural disorder and weak interactions, pose significant challenges for structural characterization by conventional methods, which typically rely on long-range crystalline order or strong electron scattering. Here, we report a computationally assisted polarized Raman spectroscopy strategy that enables direct, in situ determination of three-dimensional (3D) molecular structures and their intermolecular interactions, correlating molecular-scale orientations with macroscopic morphologies. We validated our approach using the well-characterized α-glycine crystal, achieving structural precision comparable to that of single-crystal X-ray diffraction (RMSD = 0.367 ± 0.003 Å). Extending our method to structurally complex peptides, we resolve biomaterial diphenylalanine structure (RMSD = 0.743 ± 0.009 Å) and determine Alzheimer's-associated peptide (r-acAβ) assemblies at angstrom precision. Furthermore, our approach uniquely identified specific vibrational signatures associated with hydrogen bonding and hydrophobic interactions, linking the reversible switching of peptide self-assembly through environmental stimuli. This method provides a transformative structural characterization platform, offering critical insights into molecular assembly mechanisms and enabling the rational design of next-generation functional biomaterials.