The common assumption that precision is the limit of accuracy in localizationmicroscopy and the typical absence of comprehensive calibration of opticalmicroscopes lead to a widespread issue - overconfidence in measurement resultswith nanoscale statistical uncertainties that can be invalid due to microscalesystematic errors. In this article, we report a comprehensive solution to thisunderappreciated problem of false precision. We develop arrays of subresolutionapertures as the first reference materials that enable subnanometerlocalization accuracy in widefield optical microscopy. We present novel methodsto calibrate our microscope system using aperture arrays, introducing thelight-weighting objective function for robust localization of point sourceswith fitting errors from optical aberrations, revealing the surprisingpotential utility of signal saturation, and developing an aberration correctionthat reaches the limit of fabrication precision of our reference material. In afirst application of our new measurement capability, we introduce criticaldimension localization microscopy, facilitating tests of nanofabricationprocesses and quality control of aperture arrays. In a second application, weapply these stable reference materials to answer open questions about theapparent instability of fluorescent nanoparticles that commonly serve asfiducial markers. Our study establishes a foundation for correct quantificationof measurement results in localization microscopy.
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