SIMworks-an integrated software suite for quality-controlled augmented quantitative structured illumination microscopy.

Super-resolution structured illumination microscopy (SIM) is a powerful and versatile technique for investigating fundamental cell biological processes underlying development and disease. With the growing availability of commercial SIM systems, the emergence of advanced SIM modalities and increased access through imaging facilities, adoption of SIM across research laboratories is accelerating. However, fully leveraging the capabilities of SIM remains challenging, even for experienced microscopists, due to its susceptibility to imaging artifacts and the limited availability of accessible analysis pipelines. Here, to address these challenges, we developed SIMworks, a broadly compatible, integrated software platform designed to streamline quality control, processing and quantitative analysis of large-scale SIM datasets. Building on our previously established tools-SIMcheck, Chromagnon, ChaiN and SIMinspector-SIMworks provides a unified framework compatible with both custom and commercial SIM systems and modalities. Its modular architecture includes tools for calibration, alignment and data processing, as well as robust artifact detection and correction. These are complemented by segmentation and quantification modules for a broad range of cellular and nuclear structures, enabling automated, intensity-based analysis. Each module includes detailed guidance on interpretation and troubleshooting to support usability and reproducibility, enabling both new learners and experienced users to apply best practices in SIM imaging. The complete workflow can be performed within approximately half a day, depending on dataset size and complexity. SIMworks represents the first comprehensive, modular solution for SIM data processing, augmentation and quantitative analysis, lowering technical barriers and enhancing data reliability. By facilitating rigorous workflows, SIMworks promotes the broader integration of quantitative SIM imaging into biomedical research.