A Colour-Encoded Nanometric Ruler for Axial Super-Resolution Microscopies

Recent progress has boosted the resolving power of optical microscopies to spatial dimensions well below the diffraction limit. Yet, axial super-resolution and axial single-molecule localisation typically require more complicated implementations than their lateral counterparts. In the present work, we propose a simple solution for axial metrology by providing a multi-layered single-excitation, dual-emission test slide, in which axial distance is colour-encoded. Our test slide combines on a standard microscope coverslip substrate two flat, thin, uniform and brightly emitting fluorophore layers, separated by a nanometric transparent spacer layer having a refractive index close to a biological cell. The ensemble is sealed in an index-matched protective polymer. As a proof-of-principle, we estimate the light confinement resulting from evanescent-wave excitation in total internal reflection fluorescence (TIRF) microscopy. Our test sample permits, even for the non-expert user, a facile axial metrology at the sub-100-nm scale, a critical requirement for axial super-resolution, as well as near-surface imaging, spectroscopy and sensing.