Single-Particle Photoluminescence Measures a Heterogeneous Distribution of Differential Circular Absorbance of Gold Nanoparticle Aggregates near Constricted Thioflavin T Molecules.

The chirality of biomacromolecules is critical for their function, but the optical signal of this chirality is small in the visible range. Plasmonic nanoparticles are antennas that can couple to this chiral signal. Here, we examine the molecular-scale mechanism behind the induced circular dichroism of gold nanorods (AuNRs) in solution with insulin fibrils and the fibril-intercalating dye thioflavin T (ThT) with polarization-resolved single-molecule fluorescence and single-particle photoluminescence (PL) imaging. We compared the PL upon excitation by left- and right-handed circularly polarized light to calculate the differential absorbance of AuNRs near insulin fibrils with and without ThT. Overall, our results indicate that AuNRs do not act as chiral absorbers near constricted ThT molecules. Instead, we hypothesize that fibrils promote AuNR aggregation, and this templating is mediated by subtle changes in the solution conditions; under the right conditions, only a few chiral aggregates with significantly higher circular dichroism signal contribute to a large net circular dichroism.

Single Gold Nanobipyramids Sensing the Chirality of Amyloids.

Plasmonic nanoparticles, due to their sensitivity to small changes in their closest environment and plasmon resonance, can sense the chirality of the surrounding molecules. Therefore, plasmonic nanoparticles can be applied as a next-generation biosensor for peptides or proteins. In this work, we explore the interaction between chiral, ordered protein aggregates (amyloids) and small gold nanobipyramids. We show how the morphology, structure, and chiroptical properties of amyloids induce circular dichroism in the plasmon resonance wavelengths from individual plasmonic nanoparticles upon binding to the chiral amyloid template. Moreover, using the data from microscopic and spectroscopic analyses of formed heterostructures, we propose the most probable mechanism behind the induction of chirality in this system and discuss which specific feature of insulin protein aggregates is sensed by nanobipyramids.

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 penetration, efficient operation in densely packed systems, and reduced angular photoselection of fluorophores. Thus, the introduction of polarization analysis in two-photon fluorescence microscopy (2PFM) provides a more precise determination of molecular organization in a sample compared to standard imaging methods based on linear optical processes. In this work, we focus on polarization-sensitive 2PFM (ps-2PFM) and its application in the determination of molecular ordering within complex bio-structures-amyloid spherulites. Neurodegenerative diseases such as Alzheimer's or Parkinson's are often diagnosed through the detection of amyloids-protein aggregates formed due to an impaired protein misfolding process. Exploring their structure leads to a better understanding of their creation pathway and consequently, to developing more sensitive diagnostic methods. This paper presents the ps-2PFM adapted for the determination of local fibril ordering inside the bovine insulin spherulites and spherical amyloidogenic protein aggregates. Moreover, we prove that the proposed technique can resolve the three-dimensional organization of fibrils inside the spherulite.

One- and Two-Photon Excited Autofluorescence of Lysozyme Amyloids.

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.

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 de novo 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, i.e. 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.

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

Amyloids are broadly investigated protein misfolding products with characteristic ß-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.