Fluorochromes

Fluorescence results from a process that occurs when certain molecules called fluorophores, fluorochromes, or fluorescent dyes absorb light. The absorption of light by a population of these molecules raises their energy level to a brief excited state. As they decay from this excited state, they emit fluorescent light.

When a photon, supplied by an external source such as a lamp or a laser, is absorbed by a fluorophore, this creates an excited, unstable, electronic state (S1). This process distinguishes fluorescence from bioluminescence, in which the excited state is created by a chemical reaction e.g. aequorin, luciferase. The excited state of a fluorophore is characterised by a very short half-life, usually of the order of a few nanoseconds. During this brief period, the excited molecules generally relax toward the lowest vibrational energy level within the electronic excited state. The energy lost in this relaxation is dissipated as heat. It is from the resulting relaxed singlet excited state (S1) that fluorescence emission originates. When a fluorochrome molecule falls from the excited state to the ground state, light is often emitted at a characteristic wavelength. The energy of the emitted photon is the difference between the energy levels of the two states, and that energy difference determines the wavelength of the emitted light.