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Today, refined single\u2010molecule localization microscopy methods achieve spatial resolutions in the one\u2010digit nanometer range. As the race for molecular resolution fluorescence imaging with visible light continues, reliable biologically compatible reference structures will become essential to validate the resolution power. Here, PicoRulers (protein\u2010based imaging calibration optical rulers), multilabeled oligomeric proteins designed as advanced molecular nanorulers for super\u2010resolution fluorescence imaging are introduced. Genetic code expansion (GCE) is used to site\u2010specifically incorporate three noncanonical amino acids (ncAAs) into the homotrimeric proliferating cell nuclear antigen (PCNA) at 6\u00a0nm distances. Bioorthogonal click labeling with tetrazine\u2010dyes and tetrazine\u2010functionalized oligonucleotides allows efficient labeling of the PicoRuler with minimal linkage error. Time\u2010resolved photoswitching fingerprint analysis is used to demonstrate the successful synthesis and DNA\u2010based points accumulation for imaging in nanoscale topography (DNA\u2010PAINT) is used to resolve 6\u00a0nm PCNA PicoRulers. Since PicoRulers maintain their structural integrity under cellular conditions they represent ideal molecular nanorulers for benchmarking the performance of super\u2010resolution imaging techniques, particularly in complex biological environments.<\/jats:p>","DOI":"10.1002\/adma.202310104","type":"journal-article","created":{"date-parts":[[2023,11,27]],"date-time":"2023-11-27T18:29:16Z","timestamp":1701109756000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["PCNA as Protein\u2010Based Nanoruler for Sub\u201010\u00a0nm Fluorescence Imaging"],"prefix":"10.1002","volume":"36","author":[{"given":"Dominic A.","family":"Helmerich","sequence":"first","affiliation":[{"name":"Department of Biotechnology and Biophysics, Biocenter University of W\u00fcrzburg  Am Hubland 97074 W\u00fcrzburg 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