We present an approach for fabrication of reproducible, chemically and mechanically robust functionalized layers based on MgF2 thin films on thin glass substrates. These show great advantages for use in super-resolution microscopy as well as for multi-electrode-array fabrication and are especially suited for combination of these techniques. The transparency of the coated substrates with the low refractive index material is adjustable by the layer thickness and can be increased above 92%. Due to the hydrophobic and lipophilic properties of the thin crystalline MgF2 layers, the temporal stable adhesion needed for fixation of thin tissue, e.g. cryogenic brain slices is given. This has been tested using localization-based super-resolution microscopy with currently highest spatial resolution in light microscopy. We demonstrated that direct stochastic optical reconstruction microscopy revealed in reliable imaging of structures of central synapses by use of double immunostaining of post- (homer1 and GluA2) and presynaptic (bassoon) marker structure in a 10 µm brain slice without additional fixing of the slices. Due to the proven additional electrical insulating effect of MgF2 layers, surfaces of multi-electrode-arrays were coated with this material and tested by voltage-current-measurements. MgF2 coated multi-electrode-arrays can be used as a functionalized microscope cover slip for combination with live-cell super-resolution microscopy.