A lighting device for total-internal-reflection fluorescence microscopy includes a substrate that is transparent to light, having a refractive index higher than that of water; a light-emitting device arranged in the interior of the substrate, suitable for emitting light radiation in the direction of a surface of the substrate, the light-emitting device being arranged such that at least one portion of the radiation reaches the surface with an angle of incidence larger than or equal to a critical angle of total internal reflection for an interface between the substrate and water; and at least one opaque mask, arranged in the interior or on the surface of the substrate so as to intercept a portion of the radiation that, in the absence of the mask, would reach the surface with an angle of incidence smaller than the critical angle. A lighting device to total-internal-reflection fluorescence microscopy is provided.