Programmable Protein Reference Standards for benchmarking sub-10 nm Fluorescence Microscopy

Fluorescence microscopy is increasingly used to measure molecular organization at length scales where labeling, photophysics and sample preparation can dominate quantitative accuracy. Reference standards are therefore needed that combine defined nanoscale geometry with a protein-like environment and compatibility with biological imaging. Here we introduce circular tandem repeat protein as programmable protein standards for benchmarking sub-10-nm fluorescence microscopy. Using genetic code expansion and bioorthogonal labeling, we generated compact protein rings carrying up to six labeling sites. Photoswitching fingerprint analysis revealed geometry-dependent localization accumulation and blinking kinetics, demonstrating that short-range fluorophore interactions can be assessed as measurable benchmark parameters. DNA-PAINT confirmed accessible docking sites and programmed valency at the single-particle level. We further established recombinant tethering and genetically encoded membrane display, extending the standards to cellular environments. cTRP PicoRulers were also compatible with expansion microscopy. Together, cTRPs provide a modular protein-based platform for evaluating molecular-scale imaging performance in purified and cellular environments.