2D polarization imaging as a low-cost fluorescence method to detect α-synuclein aggregation ex vivo in models of Parkinson’s disease

Abstract

A hallmark of Parkinson’s disease is the formation of large protein-rich aggregates in neurons, where α-synuclein is the most abundant protein. A standard approach to visualize aggregation is to fluorescently label the proteins of interest. Then, highly fluorescent regions are assumed to contain aggregated proteins. However, fluorescence brightness alone cannot discriminate micrometer-sized regions with high expression of non-aggregated proteins from regions where the proteins are aggregated on the molecular scale. Here, we demonstrate that 2-dimensional polarization imaging can discriminate between preformed non-aggregated and aggregated forms of α-synuclein, and detect increased aggregation in brain tissues of transgenic mice. This imaging method assesses homo-FRET between labels by measuring fluorescence polarization in excitation and emission simultaneously, which translates into higher contrast than fluorescence anisotropy imaging. Exploring earlier aggregation states of α-synuclein using such technically simple imaging method could lead to crucial improvements in our understanding of α-synuclein-mediated pathology in Parkinson’s Disease.

Excitation Energy Migration Unveils Fuzzy Interfaces within the Amyloid Architecture

“…Such energy migration events between the different randomly oriented identical fluorophores positioned within the Förster distance depolarize the fluorescence anisotropy because of the apparent orientational randomization independent of rotational dynamics. Such a depolarization via homo-FRET has been previously observed in a-syn amyloid fibrils (30)(31)(32). The energy migration efficiency measured by the observed loss in the fluorescence anisotropy at a given local density of fluorophore is related to the interdye distance.…”

Polarization-Encoded Colocalization Microscopy at Cryogenic Temperatures

“…We have shown that including the polarization degree of freedom in the localization analysis allows for more robust assignment of fluorescence photons to individual emitters. Aside from boosting the localization accuracy, this approach also provides direct access to molecular orientations which can be useful in the context of studying agglomeration or oligomerization of proteins. − Furthermore, we have shown that polarization can be used to achieve controllable switching, which is largely independent of the stochastic blinking, works with lower light levels and allows for longer camera integration times. The implementations of the ideas in our work are straightforward and not restricted to specific photophysical properties.…”

Influence of nanobody binding on fluorescence emission, mobility, and organization of GFP-tagged proteins

“…Lack of co-diffusion suggests that this complex is not strongly fluorescent (e.g., due to self-quenching) but still primarily recognized by the Nb. Fluorescence modulation upon aggregation or mis-folding of FPs and their chromophores has been described before (Camacho et al, 2018;Ge et al, 2017;Jung et al, 2005b;Kruitwagen et al, 2015;Stepanenko et al, 2008). Upon binding, Nb could partially disassemble the dark complex and release a few molecules from the complex that become fluorescent but are not bound to a Nb.…”