Open-source Sub-Nanometer Stabilization System for Super-resolution Fluorescence Microscopy

Recent advances in fluorescence nanoscopy have pushed resolution to the 1-10 nm range, enabling the direct visualization of individual molecules even in crowded biological environments. Achieving this level of precision requires rigorous sample drift control. Techniques such as MINFLUX and RASTMIN, which rely on keeping the sample fixed within an excitation pattern, demand active drift correction to achieve their theoretical nanometer-scale resolution limits. Here, we present an active stabilization system for super-resolution microscopy that delivers sub-nm precision for hours. Featuring a simple optical design, the system can be added as a separate module to any fluorescence microscope. We also provide an open-source control software including a user-friendly graphical interface readily adaptable to different setups. We demonstrate the adaptability and performance of the stabilization system with p-MINFLUX and RASTMIN measurements performed in two different setups reaching the theoretical Cramér-Rao Bound and resolving ~10 nm distances within DNA origami structures.