polarization-sensitive-two-photon-microscopy-for-label-free-amyloid

Polarization-Sensitive Two-Photon Microscopy for a Label-Free Amyloid Structural Characterization

Compared to its one-photon counterpart, two-photon excitation is beneficial for bioimaging experiments because of its lower phototoxicity, deeper tissue ...

Wrocław University of Science and Technology

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.

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

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.

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

Autofluorescence properties of amyloid fibrils are of much interest but, to date, the attention has been given mostly to one-photon excited fluorescence (1PEF), while the two-photon excited fluorescence (2PEF) properties of amyloids are much less explored. We investigate 1PEF and 2PEF of hen egg-white lysozyme (HEWL) in the form of monomers and fibrils. HEWL monomers feature some autofluorescence, which is enhanced in the case of fibrils. Moreover, by varying NaCl content, we introduce changes to fibrils morphology and show how the increase of the salt concentration is linked with an increase of 1PEF and 2PEF intensities. Interestingly, we observe 2PEF emission red-shifted in comparison to 1PEF. We confirm the presence of different relaxation pathways upon one- or two-photon excitation by different lifetimes of the fluorescence decays. Finally, we correlate the changes in optical properties of HEWL fibrils and monomers with salt-mediated changes in their morphology and the secondary structure.

Article	Year
Two-Photon Excited Polarization-Dependent Autofluorescence of Amyloids as a Label-Free Method of Fibril Organization Imaging. The journal of physical chemistry letters\| PubMed ID: 33522819	2021
Amyloid fibrils in superstructures - local ordering revealed by polarization analysis of two-photon excited autofluorescence. Biomaterials science\| PubMed ID: 35175263	2022
One- and Two-Photon Excited Autofluorescence of Lysozyme Amyloids. The journal of physical chemistry letters\| PubMed ID: 35605187	2022

Maciej Lipok

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