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Fluorescence photons emitted by single molecules contain rich information regarding their rotational motions, but adapting single-molecule localization microscopy (SMLM) to measure their orientations and rotational mobilities with high precision remains a challenge. Inspired by dipole radiation patterns, we design and implement a Tri-spot point spread function (PSF) that simultaneously measures the three-dimensional orientation and the rotational mobility of dipole-like emitters across a large field of view. We show that the orientation measurements done using the Tri-spot PSF are sufficiently accurate to correct the anisotropy-based localization bias, from 30 nm to 7 nm, in SMLM. We further characterize the emission anisotropy of fluorescent beads, revealing that both 20-nm and 100-nm diameter beads emit light significantly differently from isotropic point sources. Exciting 100-nm beads with linearly polarized light, we observe significant depolarization of the emitted fluorescence using the Tri-spot PSF that is difficult to detect using other methods. Finally, we demonstrate that the Tri-spot PSF detects rotational dynamics of single molecules within a polymer thin film that are not observable by conventional SMLM.

Imaging the three-dimensional orientation and rotational mobility of fluorescent emitters using the Tri-spot point spread function