NanoDriftGuard: Open-source isotropic ångström-scale active stabilization for super-resolution microscopy

Single-molecule localization microscopy (SMLM) achieves nanoscale resolution but remains vulnerable to sample drift, which can compromise data quality. Widely used drift correction methods, based on either fluorescent fiducials or localization events, each have inherent limitations. Here, we present a complete sample stabilization solution combining an imaging system for detecting fiducial markers and NanoDriftGuard, a real-time drift correction software. This open-source software package is available on GitHub (https://github.com/ZhengyiZ/NanoDriftGuard). This approach can achieve isotropic three-dimensional (3D) stability with a precision of ∼4 Å. Our MATLAB-based implementation is optimized for rapid execution, allowing real-time drift correction at a rate exceeding 50 Hz. The system requires only a single laser for 3D drift correction and features a compact design that minimizes residual drift to ∼10 nm/h, ensuring exceptional long-term stability. Finally, we demonstrate the system's performance by imaging nuclear pore complexes using SMLM.