A fluorescence microscope is a conventional compound microscope that has been equipped with a high-intensity light source (usually a mercury arc lamp) that emits light in a broad spectrum from visible through ultraviolet. Most conventional fluorescence microscopes utilize incident illumination to illuminate the sample from above. In this way the objective lens is used as both the illumination condenser and the fluorescent light collector. The arrangement of optical components that permits illumination from above the specimen is termed epifluorescence illumination, epi-illumination, or reflected light illumination.

The wavelength of illumination (excitation: Ex) is chosen by placing a filter after the light source that limits light transmission to a narrow range of wavelengths. The light then impinges on a dichroic mirror (beam splitter) and is reflected down through the objective lens and onto the sample. Pigment molecules within the specimen (either intrinsic or applied) absorb the light and reirradiate the energy at a longer wavelength — this is fluorescence. The objective lens collects this emitted fluorescent light (emission: Em), which then passes through the dichroic mirror. Any reflected excitation light is blocked by a third filter (barrier filter). Thus, only light emitted from fluorescent molecules within the specimen are observed or photographed. This arrangement of three optical filters is referred to as a  fluorescence cube, since they are almost always mounted together in a cubic metal mount. The user selects different wavelengths by selecting different filter set cubes. The mechanical arrangement may be either a sliding or rotating filter cube holder.

1: Ex filter
2: Dichroic mirror
3: Em (barrier) filter