Nanorulers for Super-Resolution Microscopy.

Super-resolution fluorescence microscopy has advanced into a regime where molecular-scale organization can be resolved with nanometer precision. In this context, the accurate interpretation of imaging data increasingly depends on well-defined calibration standards. Nanorulers have emerged as essential tools to validate resolution, quantify labeling performance, and benchmark analytical methods. Current approaches include DNA-based, protein-based, and virus-based nanorulers, each offering distinct advantages in terms of programmability, structural definition, and biological relevance. This minireview provides an overview of these complementary strategies, with a particular focus on recent developments in protein-based nanorulers enabled by advances in protein engineering and site-specific labeling. Together, these approaches contribute to establishing reliable calibration frameworks for quantitative molecular bioimaging.