Two-Photon Excited Polarization-Dependent Autofluorescence of Amyloids as a Label-Free Method of Fibril Organization Imaging

[Image: see text] Amyloids are broadly investigated protein misfolding products with characteristic β-sheet assemblies that have an important role in neurodegenerative diseases (e.g., Alzheimer’s disease). While they are usually visualized by staining with Thioflavin-T, Congo Red, or other fluorescent markers, it still arouses a controversy over possible staining molecule influence on the amyloid structure or aggregation process. In this work we present, for the first time, the polarization analysis of two-photon excited autofluorescence of amyloids and confirm that polarization dependence of the observed emission can be correlated with the orientation of fibrils. We show the potential of two-photon excited autofluorescence for resolution of molecular organization of fibrils within amyloid superstructures. This label-free method is compatible with two-photon imaging already applied in investigation of neurodegeneration model in mice.