Molecules are capable of absorbing photons, thus becoming electronically excited. A molecule’s return to its “normal” (ground ) state followed by the emission of light by a molecule is called fluorescence. The absorption and fluorescence effects are determined by the energy level structure of the electrons in a molecule, and thus they are specific for each individual type of molecule (see details in electron-vibrational spectroscopy).

Biological materials are generally characterised by their rather low fluorescence, but the use of diverse bright fluorescent molecules (fluorophores) that can specifically color various structures of tissues and cells made fluorescence microscopy a valuable technique for medical and biological science.

Traditional fluorescence microscopy methods offer a much lower resolution than electron microscopy or atomic force microscopy. In contrast, optical microscopy makes it possible to observe the inner microstructure of cells and even small organisms, notably both living and fixed. This makes fluorescence microscopy the optimal method for studying organism functioning on cellular, subcellular and molecular levels.

A fluorescence microscope consists of a source of light used to excite fluorophores, a detector that records emission from fluorophores, and an optical system to focus light and magnify a specimen. Ernst Abbe wrote that the resolution of an optical system built with lenses is limited by light diffraction. The maximum distance at which two objects can be distinguished (d) depends on the wavelength of light , objective’s aperture angle and the refractive index of a medium (n): .