Brilliant fluorescence signals with virtually no background noise or cross talk are the goal of FISH analysis in imaging systems. The precise selection of hardware components such as optical filters and light sources plays an important role in this. Taking the properties of fluorescent dyes into account is the basis for configuring perfectly matched multicolor FISH filters (mFISH), which enable the simultaneous use of up to seven dyes. The spectral characteristics of the filters are explained here in relation to microscope configurations. Spectral cross talk, pixel shift effects, and stable energy output are the main problems encountered in daily work. Special hard-coated single-band filters with low bandwidth but maximum transmission avoid cross talk to a high degree. The use of multiband filters allows the simultaneous imaging of up to four dyes, and multiband systems with separate exciters and emitters in filter wheels can be controlled by software. A comparison of mercury and metal halide light sources with new light sources based on light-emitting diodes (LEDs) shows the possibility of stable and long-lasting light output from the new LED light sources. White light LED light sources are now the perfect choice as a replacement for mercury-containing lamps. The combination of hard-coated mFISH filters with stable LED light sources is a very helpful tool in everyday work.