Calibration standard for evanescence microscopy

A calibration standard for determining an evanescent field axial intensity decay related to an evanescent field generated close to the interface between a sample to be tested and a substrate on which said sample is to be deposited, said calibration standard comprising one layered device stacked on a dielectric substrate, the interface between said dielectric substrate and said layered device mimicking an interface between the dielectric substrate and a sample to be tested, the layered device presents on said dielectric substrate, a surface S, and comprises:

• a series of layered structures stacked on the dielectric substrate, one above to the other,
• each layered structure comprising:
• one spacer layer, and one fluorescent emitter layer containing at least one fluorescent emitter material;
• said dielectric substrate is of a higher refractive index than the spacer layers;
• a first spacer layer being in contact with the said dielectric substrate, to present a flat surface on which the stack of the layered structures is deposited;
• a final emitter layer being covered with a final protective layer, wherein:
• each layer having a constant thickness over the surface S;
• each fluorescent emitter layer is arranged at a fixed controlled axial distance of said interface,
• each fluorescent emitter layer comprising at least one fluorescent emitter material having at least one fluorescence parameter different from the fluorescent emitter material of another fluorescent emitter layer, emission wavelengths, emission directionality, emission polarizabilities or fluorescence lifetimes,
• so that each fluorescent emitter layer having a specific fluorescence parameter at a fixed controlled axial distance of the interface, at a common excitation wavelength,
• to determine the evanescent field axial intensity decay from the combination of these specific fluorescence parameters measured at different controlled axial distances of the interface.