Image-based 3D active sample stabilization on the nanometer scale for optical microscopy

Super-resolution microscopy often entails long acquisition times of minutes to hours. Since drifts during the acquisition adversely affect data quality, active sample stabilization is commonly used for some of these techniques to reach their full potential. While drifts in the lateral plane can often be corrected after acquisition, this is not always possible or may come with drawbacks. Therefore, it is appealing to stabilize sample position in three dimensions during acquisition. Various schemes for active sample stabilization have been demonstrated previously, with some reaching sub-nm stability in three dimensions. However, these high-performance implementations significantly added to the complexity of the hardware and/or sample preparation. Here, we present a scheme for active drift correction that delivers the nm-scale 3D stability demanded by state-of-the-art super-resolution techniques and is straightforward to implement. Using a refined algorithm that does not depend on sparse peaks typically provided by fiducial markers added to the sample, we stabilized our sample position to [~]1 nm in 3D using objective lenses both with high and low numerical aperture. Our implementation requires only the addition of a standard widefield imaging path and we provide an open-source control software with graphical user interface to facilitate easy adoption of the module. Finally, we demonstrate how this has the potential to enhance data collection for diffraction-limited and super-resolution imaging techniques using single-molecule localization microscopy and cryo-confocal imaging as showcases. Why it mattersSuper-resolution light microscopy has enabled the visualization of biological structures down to the nm-scale. However, uncorrected drifts during often extended acquisition times may adversely affect data quality. Active drift correction in three dimensions has achieved sub-nm stabilization, but state-of-the-art techniques come with considerable overhead on sample preparation and/or hardware. Here, we demonstrate an image-based stabilization scheme which allows for flexibility regarding structures used for stabilization and is straightforward to adopt. Using a maximally simple implementation, we stabilized the position of our sample to around 1 nm over extended acquisition times and demonstrated usefulness in two example imaging settings where sample drifts are critical, super-resolution single-molecule localization microscopy and confocal imaging at cryogenic temperatures.