Separation estimation of two freely rotating dipole emitters near the quantum limit

According to Rayleigh’s criterion, two incoherent emitters with a separation below the diffraction limit are not resolvable with a conventional fluorescence microscope. One method of Super-Resolution Microscopy (SRM) circumvents the diffraction-limited resolution by precisely estimating the position of spatiotemporally independent emitters. However, these methods of SRM techniques are not optimal for estimating the separation of two simultaneously excited emitters. Recently, a number of detection methods based on modal imaging have been developed to achieve the quantum Cramér-Rao lower bound (QCRB) to estimate the separations between two nearby emitters. The QCRB determines the minimum achievable precision for all possible detection methods. Current modal imaging techniques assume a scalar field generated from a point source, such as a distant source from an optical fiber or a pinhole. However, for fluorescently labeled samples, point emitters are single fluorophores that are modeled as dipole emitters and, in practice, are often freely rotating. Dipole radiation must be described by vectorial theory, and the assumption of a scalar field no longer holds. Here, we present a method to numerically calculate the QCRB for measuring the separation of two dipole emitters, incorporating the vectorial theory. Furthermore, we propose a near-quantum optimal detection scheme based on one of the modal imaging techniques, super-localization by image inversion interferometry (SLIVER), for estimating the separation of two freely rotating dipoles. In the proposed method, we introduce a vortex wave plate before the SLIVER detection to separate the radial and azimuthal components of the dipole radiation. With numerical simulations, we demonstrated that our method achieves non-divergent precision at any separation between two dipole emitters. We investigated several practical effects relevant to experimental measurements in super-resolution microscopy, including numerical aperture, detection bandwidth, number of estimation parameters, background, and misalignment on separation estimation. Our proposed measurement provides a near quantum-limited detection scheme for measuring the separation of two freely-rotating dipole emitters, such as fluorescently tagged molecules, which are commonly used in super-resolution microscopy.