Traceable localization in optical microscopy

Localization microscopy enables resolution beyond the limit of optical diffraction, engendering many opportunities across the physical and life sciences. With enough signal photons, the localization precision of sparse images can extend into the subnanometer scale. Supporting accuracy is challenging, however, as systematic errors can be orders of magnitude larger across an imaging field. To solve this critical but often ignored problem, we are developing arrays of nanoscale apertures into traceable standards for localization microscopy. We fabricate aperture arrays by electron-beam lithography and measure aperture positions by critical-dimension atomic-force microscopy. Correlative measurements by optical microscopy reveal localization errors due to optical aberrations, which we correct by a Zernike model. Statistical analysis of aperture positions correlates surface structure and optical transmission to within a few nanometers. Our calibration establishes the new concept of a localization uncertainty field, with localization errors and scale uncertainty yielding regions of position traceability to within a 68 % coverage interval of ± 1.0 nm. In this way, our study achieves new traceability in localization microscopy, enabling reliable position data for meaningful comparison.