Fabrication of versatile calibration samples for 3D super-resolution optical imaging with <5nm Z resolution using greyscale e-beam lithography and Nano-Imprint replication

We present a 3D mold fabrication process to produce high precision calibration samples with few nanometers steps in the vertical direction. After imprinting into a resist layer of adequate refractive index using nano-imprint lithography, these molds can produce calibration samples valuable for super-resolution Direct Optical Nanoscopy with Axially Localized Detection (DONALD). This novel method is dedicated to the 3D mapping of labeled biomolecules into biological samples by fluorescence and requires calibration samples with well-characterized steps ranging from 5 nm to 125 nm in height.