Embodiments of the present invention can resolve molecules beyond the optical diffraction limit in three dimensions. A double-helix point spread function can be used to in conjunction with a microscope to provide dual-lobed images of a molecule. Based on the rotation of the dual-lobed image, the axial position of the molecule can be estimated or determined. In some embodiments, the angular rotation of the dual-lobed imaged can be determined using a centroid fit calculation or by finding the midpoints of the centers of the two lobes. Regardless of the technique, the correspondence between the rotation and axial position can be utilized. A double-helix point spread function can also be used to determine the lateral positions of molecules and hence their three-dimensional location.