Layer-by-layer technique is used to adsorb a uniform ultrathin layer of fluorescently labelled polyelectrolytes on a glass cover slip. Due to their thickness, uniformity and fluorescence properties, these ultrathin layers may serve as a simple and applicable standard to directly measure the z-response of different scanning optical microscopes. In this work we use ultrathin layers to measure the z-response of confocal, two-photon excitation and 4Pi laser scanning microscopes. Moreover, due to their uniformity over a wide region, i.e. cover slip surface, it is possible to quantify the z-response of the system over a full field of view area. This property, coupled with a bright fluorescence signal, enables the use of polyelectrolyte layers for representation on sectioned imaging property charts: a very powerful method to characterize image formation properties and capabilities (z-response, off-axis aberration, spherical aberration, etc.) of a three-dimensional scanning system. The sectioned imaging property charts method needs a through-focus dataset taken from such ultrathin layers. Using a comparatively low illumination no significant bleaching occurs during the excitation process, so it is possible to achieve long-term monitoring of the z-response of the system. All the above mentioned properties make such ultrathin layers a suitable candidate for calibration and a powerful tool for real-time evaluation of the optical sectioning capabilities of different three-dimensional scanning systems especially when coupled to sectioned imaging property charts.