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bioRxiv · 10.1101/2020.01.22.916122

Single-molecule orientation localization microscopy for resolving structural heterogeneities within amyloid fibrils

Ding, T.·Wu, T.·Mazidi, H.·Zhang, O.·Lew, M. D.
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Abstract

Simultaneous measurements of single-molecule positions and orientations provide critical insight into a variety of biological and chemical processes. Various engineered point spread functions (PSFs) have been introduced for measuring the orientation and rotational diffusion of dipole-like emitters, but the widely used Cramer-Rao bound (CRB) only evaluates performance for one specific orientation at a time. Here, we report a performance metric, termed variance upper bound (VUB), that yields a global maximum CRB for all possible molecular orientations, thereby enabling the measurement performance of any PSF to be computed efficiently (~1000x faster than calculating average CRB). Our VUB reveals that the simple polarized standard PSF provides robust and precise orientation measurements if emitters are near a refractive index interface. Using this PSF, we measure the orientations and positions of Nile red (NR) molecules transiently bound to amyloid aggregates. Our super-resolved images reveal the main binding mode of NR on amyloid fiber surfaces, as well as structural heterogeneities along amyloid fibrillar networks, that cannot be resolved by single-molecule localization alone.

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Ding, T., Wu, T., Mazidi, H., Zhang, O., Lew, M. D.. 2020-01-23. Single-molecule orientation localization microscopy for resolving structural heterogeneities within amyloid fibrils. https://doi.org/10.1101/2020.01.22.916122

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