3.1 Fluorescence is activated by light

What makes fluorescent molecules like GFP ‘glow’ the way they do? To understand this, it is necessary to look at the molecular level and specifically at the arrangements of atoms. Atoms contain a central nucleus around which electrons move in electron shells (or orbitals). The energy state of electrons increases from the innermost to the outer shells.

Fluorescent molecules, also called fluorophores, generally contain rings of carbon atoms (called aromatic rings). These molecules can absorb incoming energy (usually in the form of light). As a result of the absorption of energy, electrons within the molecules change from a resting condition called the ‘ground state’ to an ‘excited state’ on a shell further away from the nucleus for a very brief period of time. Eventually, the excited electrons return to their ground state, and as they do this, they emit some of the energy they had originally absorbed as photons of light (Figure 9).

Figure 9 A single atom within a molecule is shown absorbing energy, which excites an electron from its ground state to an excited state. When the electron returns to its ground state, light is emitted.

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Three atoms are illustrated in the diagram, each with a central nucleus (brown circle) surrounded by two rings. From left to right: in the ground state, an electron (blue circle) is present on the inner ring. If energy from light (indicated by the blue wavy line with an arrowhead in the middle image) is absorbed by the electron, it moves to the outer ring. From there, the electron returns to the ground state on the inner ring again (right hand side). During this process it emits fluorescent light (indicated by the red wavy line with an arrowhead).

Figure 9 A single atom within a molecule is shown absorbing energy, which excites an electron from its ground state to an excited state. ...