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 Cramér-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 (~1000× 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.
Highlights
A key strength of single-molecule localization microscopy (SMLM) is its ability to measure the full distribution of the phenomena under study and avoid ensemble averaging
Since the orientational second moment vector m is six dimensional, variance upper bound (VUB) greatly accelerates the comparison of various point spread functions (PSFs) for any specific imaging scenario
We stress that this computational speedup of measuring global performance makes VUB a useful design and optimization tool, but Cramér-Rao bound (CRB) is still useful for quantitatively characterizing measurement performance for specific molecular orientations m
Summary
A key strength of single-molecule localization microscopy (SMLM) is its ability to measure the full distribution of the phenomena under study and avoid ensemble averaging. Our VUB analysis surprisingly shows that a microscope with two polarization detection channels, exhibiting a polarized (standard) PSF, provides superior measurement precision when molecules are near a refractive index interface, especially when they lie perpendicular to the optical axis, even under low SBR. This conventional method of measuring molecular orientation has poor performance in index-matched samples unless a perturbation, such as defocus, is added to the optical system [11].
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