Enhancing precision for simultaneous 3D localization and 3D orientation with structured illumination

Determining the three-dimensional (3D) position and orientation of molecules from limited fluorescence photons is challenging. Existing modulated illumination localization microscopy partially transfers the photon burden to excitation intensity gradients, advancing precision to the molecular scale. However, most simultaneous localization and orientation methods rely on polarization splitting in the detection path and are sensitive to the signal-to-background ratio. This sensitivity is further exacerbated by sequential structured illumination (SSI), potentially compromising robustness. Here, we introduce V-SIMFLUX, which integrates SSI and the Vortex PSF without polarization-splitting detection. Our theoretical analysis shows that V-SIMFLUX improves the precision of all estimated parameters, notably achieving a typical 2.9-fold improvement in lateral localization and a 1.7-fold enhancement in azimuthal orientation compared to conventional wide-field illumination.