  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.
