Open-source 3D active sample stabilization for fluorescence microscopy.

Super-resolution microscopy has enabled imaging at nanometer-scale resolution. However, achieving this level of detail without introducing artifacts that could mislead data interpretation requires maintaining sample stability throughout the entire imaging acquisition. This process can range from a few seconds to several hours, particularly when combining live-cell imaging with super-resolution techniques. Here, we present a three-dimensional active sample stabilization system based on real-time tracking of fiducial markers. To ensure broad accessibility, the system is designed using readily available off-the-shelf optical and photonic components. Additionally, the accompanying software is open source and written in Python, facilitating adoption and customization by the community. We achieve a standard deviation of the sample movement within 1 nm in both the lateral and axial directions for a duration in the range of hours. Our approach allows easy integration into existing microscopes, not only making prolonged super-resolution microscopy more accessible but also allowing confocal and widefield live-cell imaging experiments spanning hours or even days.