The basic function of a fluorescence microscope is to irradiate the specimen with a desired and specific band of wavelengths, and then to separate the much weaker emitted fluorescence from the excitation light.

In a properly configured microscope, only the emission light should reach the eye or detector so that the resulting fluorescent structures are superimposed with high contrast against a very dark or black background when viewed in the microscope. The limits of detection in a fluorescence microscope are generally governed by the darkness of the background, and the excitation light is typically several hundred thousand to a million times brighter than the emitted fluorescence.

A modern epifluorescence microscope is equipped for both transmitted and reflected fluorescence microscopy. The vertical illuminator in the center of the diagram of a fluorescence microscope has the light source positioned at one end the episcopic lamphouse and the microscope filter cube turret at the other. The design consists of a basic reflected light microscope in which the wavelength of the reflected light is longer than that of the excitation.

Johan S. Ploem is credited with the development of the vertical illuminator for reflected light fluorescence microscope. In a fluorescence vertical illuminator of the fluorescence microscope the light of a specific wavelength or defined band of wavelengths, often in the ultraviolet, blue or green regions of the visible spectrum, is produced by passing multi-spectral light from an arc-discharge lamp or other source through a wavelength selective excitation filter. Wavelengths passed by the excitation filter reflect from the surface of a dichromatic, also termed a dichroic mirror or beam splitter, through the fluorescence microscope objective to bath the specimen with intense light. If the specimen when viewed under a fluorescence microscope fluoresces, the emission light gathered by the microscope objective passes back through the dichromatic mirror and is subsequently filtered by a barrier or emission filter, which blocks the unwanted excitation wavelengths. It is important to note that fluorescence microscope is the only mode in optical microscopy where the specimen, subsequent to excitation, produces its own light. The emitted light re-radiates spherically in all directions, regardless of the excitation light source direction.

Epi-fluorescence microscope illumination is the overwhelming choice of techniques in modern microscopy, and the reflected light vertical illuminator is interposed between the observation viewing tubes and the microscope nosepiece housing the microscope objectives. The illuminator of a fluorescence microscope is designed to direct light onto the specimen by first passing the excitation light through the microscope objective which in this configuration, acts as a condenser on the way toward the specimen, and then using that same objective to capture the emitted fluorescence. This type of fluorescence microscope illuminator has several advantages. The fluorescence microscope objective serves first as a well-corrected condenser and secondly as the image-forming light gatherer.

Being a single component, the objective or condenser of a fluorescence microscope is always in perfect alignment. A majority of the excitation light reaching the specimen when viewed under a fluorescence microscope passes through without interaction and travels away from the objective, and the illuminated area is restricted to that which is observed through the eyepieces. Unlike the situation in some microscope contrast enhancing techniques, the full numerical aperture of the microscope objective is available when the microscope is properly configured for Köhler illumination. Finally, it is possible to combine with or alternate between the reflected light fluorescence and transmitted light observation of a microscope and the capture of digital images using a microscope.