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. Also, comparison between techniques remains a major challenge due to the absence of suitable calibration tools that would permit a metrology along the microscope's optical axis. Likewise, such test samples would be useful as well for quantifying nanometric drift, or axial fluorophore mobility, or again for quantifying the sub-wavelength light confinement in near-field microscopies. In the present work, we propose a colour-encoded nanoscopic axial ruler. We provide a multi-layered single-excitation, dual-emission test slide, which translates the axial-distance problem into a spectral measurement. 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 demonstrate 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. (201 words).