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Summary: Amyloid fibrils are structurally heterogeneous protein aggregates that are implicated in a wide range of neurodegenerative including Alzheimer’s, Creutzfeldt–Jakob, Huntington’s, and Parkinson’s diseases. The secondary structure of amyloid fibrils is remarkably uniform composed of characteristic “cross-β” sheets. Here, the bound Thioflavi T in the amyloid fibril is modeled with conical distribution of dyes enabling us to estimate the fluorescence intensity in the two orthogonal polarized channels (s, p) at arbitrary fibril orientation. The model suggests that the parallel orientation of the cone axis with the s channel yields the maximum anisotropy that solely depends on the cone aperture. As a proof of principle, amyloid fibrils inspired from Aβ(1–42) have been investigated with incremental analyzer rotation to reveal orientation and angular distribution of dye. The variation of cone axis and the angular distribution is linked with the polymorphism of amyloid fibril (doi.org/10.1021/acs.jpcb.1c08604).
Fig. 2∣ Polymorphism of Aβ inspired fibrils using emission wide-field anisotropy imaging. (a) The conical angular distribution of ThT dipoles is aligned along the - polarized channel (top panel) and at an arbitrary angle (i.e., α) shown in the bottom panel. The PBS has to be rotated to align the -channel and the cone axis in the latter scenario. (b) The PBS is rotated between 0° to 180° with a 10° increment for each set of data. The (c) and the (d) cone axis orientation map from the emission anisotropy image stack. The cone orientation is represented by the semi-circle color bar, while the mean cone orientation is shown by the double-sided arrow in (d). 1 µm is the scale in (c) and (d).
We developed a method to elucidate the structural heterogeneity of amyloid fibrils based on polarization-resolved emission anisotropy imaging in wide-field microscopy. Here, the angular organization of the amyloid-specific dye thioflavin T (ThT) is modeled with open cone distribution along the fiber. The emission from the fibril-bound ThT dipoles is collected into two orthogonal polarized emission channels (S, P) using a polarizing beam splitter (PBS) kept on a rotatable mount before the camera (Fig. 2). In this configuration, fibrils oriented along the channel have predominant emissions in that channel, while some photons would spread in the orthogonal channel based on the angular distribution of ThT. A mathematical model, helical anisotropy (rh) has been proposed based on the open cone distribution of ThT to estimate the emission intensity in the S/P channels for arbitrary orientation of the fibril (Fig. 2a). The model suggests that the PBS angle where anisotropy reached a maximum (rh~rmax) corresponds to the parallel orientation of the cone axis with the S channel (Fig. 2a, left panel). Besides, it solely depends on the accessible cone aperture (θ) constrained by the twisted pitch.
We have investigated the fibrils of small peptides inspired by the biologically relevant amyloid-beta protein (Aβ1–42) while rotating the PBS with a gradual increment of S/P channel orientations (Fig. 2b). We observed strongly polarized emission from ThT bound amyloid fiber exhibiting a periodic variation with the incremental PBS rotation. From the anisotropy image stack, I have computed the rmax (Fig. 2c) and the S- channel orientation corresponding to the maximum rh-value (Fig. 2d) to extract the angular distribution of ThT and cone axis orientation at each pixel along the fibers, repectively. My analyses reveal small cone apertures for Aβ40–42 and Aβ40–41V fiber. Besides, the cone axis orientations are roughly parallel to the fiber orientation for all three samples; however, fibers with offset cone axis are also observed. I discussed the finding with the polymorphic forms of the amyloid fibril, which are challenging to study using conventional methods.
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