The popularity of fluorescence microscopy arises from the inherent mode of action, where the fluorescence emission from probes is used to visualize selected features on a presumed dark background. However, the background is rarely truly dark, and image processing and analysis is needed to enhance the fluorescent signal that is ascribed to the selected feature. The image acquisition is facilitated by using considerable illumination, bright probes at a relatively high concentration in order to make the fluorescent signal significantly more intense than the background signal. Here, we present two methods for completely removing the background signal in spectrally resolved fluorescence microscopy. The methodology is applicable for all probes with narrow and well-defined emission bands (Full width half-maximum < 20 nm). Here, we use two lanthanide based probes exploiting the narrow emission lines of europium(III) and terbium(III) ions. We used a model system with zeolites doped with lanthanides immobilized in a polymer stained with several fluorescent dyes regularly used in bioimaging. After smoothing the spectral data recorded in each pixel, they are differentiated. Method I is based on the direct sum of the gradient, while method II resolves the fluorescent signal by subtracting a background calculated via the gradient. Both methods improve signal-to-background ratio significantly and we suggest that spectral imaging of lanthanide-centered emission can be used as a tool to obtain absolute contrast in bioimaging.