The double-helix point spread function enables precise and accurate measurement of 3D single-molecule localization and orientation.

Single-molecule-based super-resolution fluorescence microscopy has recently been developed to surpass the diffraction limit by roughly an order of magnitude. These methods depend on the ability to precisely and accurately measure the position of a single-molecule emitter, typically by fitting its emission pattern to a symmetric estimator (e.g. centroid or 2D Gaussian). However, single-molecule emission patterns are not isotropic, and depend highly on the orientation of the molecule's transition dipole moment, as well as its -position. Failure to account for this fact can result in localization errors on the order of tens of nm for in-focus images, and ~50-200 nm for molecules at modest defocus. The latter range becomes especially important for three-dimensional (3D) single-molecule super-resolution techniques, which typically employ depths-of-field of up to ~2 μm. To address this issue we report the simultaneous measurement of precise and accurate 3D single-molecule position and 3D dipole orientation using the Double-Helix Point Spread Function (DH-PSF) microscope. We are thus able to significantly improve dipole-induced position errors, reducing standard deviations in lateral localization from ~2x worse than photon-limited precision (48 nm vs. 25 nm) to within 5 nm of photon-limited precision. Furthermore, by averaging many estimations of orientation we are able to improve from a lateral standard deviation of 116 nm (~4x worse than the precision, 28 nm) to 34 nm (within 6 nm).