OpenBlink: Low-Cost TIRF Microscopy for Super-Resolution Imaging via μManager

Abstract Single-molecule localization microscopy (SMLM) has become a central tool in nanoscale biological research due to its high spatial resolution and compatibility with wide-field microscopy. However, achieving quantitative SMLM requires homogeneous high-power illumination, nanometric axial stability, and precise multichannel detection, features typically found only in high-end commercial instruments or custom solutions in specialized laboratories. The cost of such microscopes and their technical complexity still limit access to these advanced imaging techniques. Several homemade single-molecule microscopes and their submodules have been demonstrated as open-source, highly customizable, and cost-effective alternatives to their commercial counterparts. Yet, implementation of such systems requires expert knowledge in optics, electronics, and control systems engineering. We introduce OpenBlink, a compact, open-source TIRF microscope that integrates powerful homogeneous quad-line laser illumination, dual-channel detection, and active focus stabilization for quantitative multicolor super-resolution imaging. OpenBlink achieves localization precision below 10 nm, supports a tunable, large field of view ranging from 105 × 105 μm2 to 210 × 210 μm2, and maintains axial stability for hours, enabling high-throughput super-resolution acquisition. Built with predominantly off-the-shelf components and full integration into open-source software μManager, where metadata registration ensures reproducibility, OpenBlink offers a low threshold for adoption by easing implementation, use, and maintenance. At a substantially reduced cost of approximately 70,000 euros, among which the high-power laser combiner alone is less than 20,000 euros, OpenBlink greatly improves accessibility for laboratories that wish to implement scalable high-performance super-resolution microscopy based on single molecules.