Asynchronous Phase Shifts are Effective for Interferometric Single‐Molecule Localization Microscopy

Abstract Interferometric techniques offer exceptional axial precision in single‐molecule 3D super‐resolution microscopy but generally rely on complex optical instrumentation to simultaneously measure multiple phase shifts of fluorescence emission. To address this limitation, here two‐step phase‐shifting interferometry (TPSI) is introduced, which utilizes asynchronous phase shifts to achieve ultra‐high interferometric axial precision with a much simpler optical setup. A theoretical framework is presented for the robustness of TPSI against intensity imbalances that arise from the stochastic blinking behaviors of fluorophores. Experimental validation demonstrates that TPSI maintains high axial precision across the entire interferometric depth, achieving 5 nm axial precision for fluorophores when ≈1500 photons are detected. TPSI‐based interferometric single‐molecule localization microscopy thus provides a streamlined and cost‐effective pathway to ultra‐high precision 3D nanoscopy.