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.