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bioRxiv · 10.1101/2025.01.10.631718

Open-Source 3D Active Sample Stabilization for Fluorescence Microscopy

Patil, S.·Vicidomini, G.·Slenders, E.
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Abstract

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 3D 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 direction 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.

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Patil, S., Vicidomini, G., Slenders, E.. 2025-01-14. Open-Source 3D Active Sample Stabilization for Fluorescence Microscopy. https://doi.org/10.1101/2025.01.10.631718

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