Whole-Mount Diffracted X-ray Blinking Analysis of Intracellular Amyloid-β-Associated Nanoscale Dynamics in Caenorhabditis elegans

Abstract Nanoscale protein dynamics are closely associated with molecular function and pathological structure formation, yet their measurement within animal tissues remains technically challenging. In this study, we developed a diffracted X-ray blinking (DXB) method to evaluate amyloid-β (Aβ)-associated nanoscale fluctuations in fixed and permeabilised whole-mount Caenorhabditis elegans. Intracellular Aβ expressed in neurons (nAβ) or body-wall muscle cells (mAβ) was labelled with gold nanoparticles using an antibody-mediated strategy, without dissection or sectioning. The labelled worms produced detectable Au(111) diffraction signals, and the resulting time-resolved diffraction intensity fluctuations were analysed by autocorrelation function (ACF) analysis. A comparison of the ACF decay constants obtained from nAβ and mAβ worms revealed that compared with nAβ, mAβ contained a greater fraction of fast relaxation components. Furthermore, in vitro DXB measurements of Aβ revealed local fast relaxation components that changed over time in association with changes in Aβ assemblies, partly resembling the Aβ-associated motional features observed in worms. These findings suggest that the detected relaxation components may reflect structurally diverse Aβ states arising during aggregation. These results demonstrate the feasibility of applying DXB to detect Aβ-associated nanoscale fluctuation signatures in fixed whole-mount C. elegans and provide a methodological framework for analysing protein-associated dynamic states in animal tissue samples.