Neurodegenerative diseases such as Parkinson’s and Alzheimer’s are accompanied by amyloid proteins that have been misfolded. However, the mechanism by which protein misfolding and aggregation trigger neurodegeneration and the identity of the neurotoxic structure is still unclear. Now researchers from Washington University in St. Louis, report they have developed a new microscopy technique that measures the location and orientation of single molecules, providing a closer look at the amyloid protein structure.

Their new technique is described in a study titled, “Single-molecule orientation localization microscopy for resolving structural heterogeneities between amyloid fibrils,” and published in Optica.

“We need imaging technologies that can watch these molecular movements in living systems to understand the fundamental biological mechanisms of disease,” stated Matthew D. Lew, leader of the research team, and assistant professor at Washington University.

The researchers developed a performance metric to characterize how sensitively various microscopes can measure orientations of fluorescent molecules. Using the new performance indicator, the researchers discovered that a microscope that splits fluorescence light into two polarization channels (x and y) provides superior and practical orientation measurements.

“By measuring the orientations of single molecules bound to amyloid aggregates, the selected microscope enabled us to map differences in amyloid structure organization that cannot be detected by standard localization microscopes,” noted Tingting Wu, a PhD student at Washington University in St. Louis and co-first author of the work.

The researchers quantified how the orientations of fluorescent molecules varied each time one attached to an amyloid protein. Differences in these binding behaviors can be attributed to structure differences between amyloid aggregates. Since the method provides single-molecule information, the researchers were able to observe nanoscale differences between amyloid structures without averaging out details of local features.

a) Single-molecule localization microscopy image of a network of amyloid aggregates. (b) Image showing Nile red binding orientations to amyloid surfaces, color-coded according to the average orientation measured within each bin. (c-g) Individual orientation measurements localized along fibril backbones within the white boxes in (b). The lines are oriented and color-coded according to the direction of the estimated angle. Horizontal white scale bars are length markers, 1 micron in (a) and (b), and 100 nm in (f) and (g). [Tianben Ding, Tingting Wu, and Matthew D. Lew, Washington University in St. Louis]
This new method opens a door of understanding for researchers and may contribute to future therapies of neurodegenerative diseases.

“We hope our single-molecule orientation imaging approach can provide new insights into amyloid structure and possibly contribute to the future development of effective therapeutics against these diseases,” said Tianben Ding, a PhD student at Washington University and co-author of the study.

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