Amyloid fibrils in superstructures - local ordering revealed by polarization analysis of two-photon excited autofluorescence.

Protein misfolding products - amyloids - tend to form distinct fibrillar structures of the characteristic fold for a given neurodegenerative disease or pathology. Moreover, amyloids (also in the intermediate or distorted state) can act as secondary nuclei for fibrillation. Such secondary nucleation amplifies plaque development correlated with various diseases. Therefore, a versatile and non-destructive method of detection and differentiation between distinct fibrillar structures is of great importance. Amyloids exhibit unique optical properties, green-blue autofluorescence, which can also be induced by two-photon excitation. Herein, we use this label-free technique to resolve local fibrillar ordering in amyloid superstructures - spherulites. With polarization-dependent two-photon excited amyloid autofluorescence, we resolved fibrillar orientation in the spherulite corona and discussed the presence of amorphous aggregates, distorted fibrils or amyloid intermediate species within the spherulite core. Our polarization sensitive two-photon microscopy investigations are supported by TEM imaging and provide a promising tool for the detection and differentiation between well-developed amyloid fibrils and amorphous/distorted structures present at different stages of the formation of amyloid superstructures and plaques.