Fluorescence is a type of photoluminescence where light raises an electron to an excited state. The excited state undergoes rapid thermal energy loss to the environment through vibrations, and then a photon is emitted from the lowest-lying singlet excited state. This process of photon emission competes for other non-radiative processes including energy transfer and heat loss. Fluorescence spectroscopy uses a beam of light that excites the electrons in molecules of certain compounds, and causes them to emit light. That light is directed towards a filter and onto a detector for measurement and identification of the molecule or changes in the molecule.

The basic function of a fluorescence spectrometer 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 configured fluorescence microscope in particular, only the emission light will reach the eye or detector so that the resulting fluorescent structures are superimposed with high contrast against a very dark background. Through the use of multiple fluorescence labelling with different probes can identify several target molecules simultaneously. 

The Fluorescence process, allows users through microscopy to determine the distribution of a single molecule species, its amount and its localization inside materials, devices, cells and co-localization with other molecules. When the reflected light and background fluorescence is filtered in this type of spectroscopy/microscopy the targeted parts of a given sample can be imaged.