Fully automated multicolour structured illumination module for super-resolution microscopy with two excitation colours

Abstract

Abstract In biological imaging, there is a demand for cost-effective, high-resolution techniques to study dynamic intracellular processes. Structured illumination microscopy (SIM) is ideal for achieving high axial and lateral resolution in live samples due to its optical sectioning and low phototoxicity. However, conventional SIM systems remain expensive and complex. We introduce openSIMMO, an open-source, fully-automated SIM module compatible with commercial microscopes, supporting dual-color excitation. Our design uses affordable single-mode fiber-coupled lasers and a digital micromirror device (DMD), integrated with the open-source ImSwitch software for real-time super-resolution imaging. This setup offers up to 1.55-fold improvement in lateral resolution over wide-field microscopy. To optimize DMD diffraction, we developed a model for tilt and roll pixel configurations, enabling use with various low-cost projectors in SIM setups. Our goal is to democratize SIM-based super-resolution microscopy by providing open-source documentation and a flexible software framework adaptable to various hardware (e.g., cameras, stages) and reconstruction algorithms, enabling more widespread super-resolution upgrades across devices.